KR20090095626A - COMPOSITIONS AND METHODS TO TREAT CANCER WITH CpG RICH DNA AND CUPREDOXINS - Google Patents

Abstract

The present invention relates to compositions comprising CpG rich DNA from Pseudomonas aeruginosa. The compositions optionally comprise a cupredoxin. The present invention includes specific CpG DNAs from Pseudomonas aeruginosa that are useful for treating cancer and other conditions in patients. These compositions are optionally in a pharmaceutically acceptable carrier and also optionally comprise a cupredoxin. The present invention further relates to methods to express proteins near cancer cells. These methods may be used to express therapeutic or diagnostic proteins near cancer cells in a patient suffering from cancer or other conditions, and can also be used for diagnosing cancer in a patient. This method uses the gene for azurin from P. aeruginosa as an expression system for azurin or heterologous proteins in P. aeruginosa or heterologous cells.

Description

COMPOSITIONS AND METHODS TO TREAT CANCER WITH CpG RICH DNA AND CUPREDOXINS}

Cross Reference to Related Applications

This application claims 35 U.S.C. U.S. filed December 4, 2006, under §§ 119 and 120. Provisional Application No. Claim priority on 60 / 872,471. The entire contents of these applications are incorporated herein by reference.

Statement of Government Rights

The gist of this application was supported by Grant Number AI 16790-21, ES 0405046, AI 45541, CA09432, N01-CM97567 from the National Institutes of Health (NIH), Bethesda, Maryland, U.S.A. The government may assert some rights in the invention.

Field of invention

The present invention relates to a composition comprising CpG rich DNA derived from Pseudomonas aeruginosa and optionally cupredoxin. The present invention includes specific CpG DNA derived from Pseudomonas aeruginosa to treat disease and prevent cancer in patients, particularly patients suffering from cancer. These compositions are optionally included in a pharmacologically acceptable carrier to optionally include cupredoxin. The invention also relates to a method of expressing a protein in the vicinity of cancer cells. Using such a method, therapeutic or diagnostic proteins may be expressed around cancer cells of patients with disorders, especially cancer patients, cancer prevention and patients diagnosed with cancer. This method uses the azurin gene derived from Pseudomonas aeruginosa as an expression system of azurin or a heterologous protein in Pseudomonas aeruginosa or dysplastic cells.

The use of live bacteria in DNA vaccination and gene therapy has shown considerable potential for cancer therapy, yet another method has used bacterial products such as polysaccharides, peptidoglycans or naked DNA. Vassaux et al., J. Pathol. 208-290-298 (2006). In early 1984, Mycobacterium bovis ) confirmed that the DNA of BCG has antitumor properties. In addition, Tokunaga et al. Described that DNA purified from BCG showed significant tumor suppression in mice and substantially suppressed human skin malignancies. Tokunaga et al, Japan J. Infect. Dis. 52: 1-11 (1999),

The immune stimulatory activity that inhibits tumors in BCG DNA is due to the presence of DNA stretch rich in unmethylated CpG dinucleotides. Non-methylated CpG sequences are more than 20 times common to bacterial DNA when compared to mammalian DNA, and the presence (motif) of certain unmethylated CpG sequences is recognized as Toll-type receptor (TLR) 9. The interaction between the bacterial CpG DNA motif and TRL9 activates single cells and dendritic cells to produce interleukin-12, which in turn activates T-helper 1 cells. In contrast to CpG DNA, bacterial lipopolysaccharide is recognized by TLR4, and the corresponding lipoprotein is recognized by TLR2. Modlin, Nature 408: 659-660 (2000); Krieg, Nature Med. 9: 831-835 (2003). Since it is difficult to purify bacterial DNA rich from non-methylated CpG sequences for human clinical trials, synthetic oligodeoxynucleotides (ODNs) of 8 to 30 bases and one or more CpGs can be used to And immunotherapy of parasitic infections as well as limited phase I human clinical trials in patients with basal cell carcinoma or melanoma. Krieg. Id.

In contrast to the bacterial CpG motif, mammalian cell DNA contains a CpG sequence, which is heavily methylated. For example, about 6-8% of human DNA cytosine residues are shown to be methylated by DNA methyltransferase, which is significantly higher in human tumor cells than normal cells. There are CpG islands in the promoter portion of many human genes, which are hypermethylated to induce silencing of downstream genes. Epigenetic silencing of such downstream genes, particularly in tumor suppressor genes such as p16Ink4a, BRCA1 or hMLH1, triggers tumor formation and the development of specific DNA demethylating substances is an anti-tumor agent. Will be used. Herman and Baylin. New Eng. J Med. 349: 2042-2054 (2003); Femberg and Tycko. Nature Rev. Cancer 4: 143-153 (2004). Transcription profiling of several breast cancer cell lines demonstrates that there are many methylated CpG islands in primary tumors and cell lines, but not in DNA of normal breast epithelium or DNA consistent with lymphoid cells of cancer patients. The treatment of breast cancer cell lines with 5-azacytidine, a DNA methylation inhibitor, inhibited the growth of cancer cells, explaining the importance of promoter CpG island methylation in tumor growth. Wang et al, Oncogene 24 2705-2714 (2005).

What is needed are new therapies for patients suffering from disorders, especially cancer patients, and prophylaxis of cancer. Such treatment may be a new treatment method or a change or amelioration of the standard described treatment. Such cancer treatments should be able to delay tumor growth in mammalian patients or to reduce tumor size in mammalian patients. There is also a need for methods of delivering therapeutic molecules to cancer cells in patients and for diagnosing the presence and location of tumors in patients.

Summary of the Invention

The present invention Pseudomonas and a composition comprising CpG rich DNA derived from aeruginosa ). In particular, these compositions may comprise CpG rich DNA derived from Pseudomonas aeruginosa and optionally a cupredoxin peptide such as the 50-77 residue portion (p28) of azurin and / or azurin of Pseudomonas aeruginosa. The invention also relates to the use of the compositions of the invention for the treatment of patients, in particular for the treatment of patients suffering from disorders, in particular for patients suffering from cancer. The present invention relates to a method of expressing a protein upon contact with cancer cells and a method of diagnosing and treating cancer by administering cells and cells.

In one aspect of the invention, an isolated nucleic acid molecule is selected from (a) a nucleic acid selected from SEQ ID NOS: 26-62, (b) a polynucleotide selected from a sequence that is at least 90% homologous to a nucleic acid selected from SEQ ID NOS: 26-62 Sequence. In certain embodiments, the polynucleotide is SEQ ID NO: 26. The isolated nucleic acid sequence may be included in a pharmacologically acceptable carrier. Such pharmaceutical compositions may comprise at least one cupredoxin peptide. In some embodiments, the pharmaceutical composition is formulated for intravenous, subcutaneous or topical administration. In some embodiments, the cupredoxin peptide in the pharmaceutical composition is Pseudomonas aeruginosa ), Alcaligenes faecalis), Achromobacter cycloalkyl class test (Achromobacter cycloclastes), Bordetella chevron kisep urticae (Bordetella bronchiseptica ), Mcthylomonas sp . , Meningococcal bacteria ( Neisseria) meningitidis ), Pseudomonas fluorescens , Pseudomonas chlorolapis Chlororaphis ), Xylella fastidiosa , and Vibrio parahaemolyticus . In some embodiments, the cupredoxin peptide included in the pharmaceutical composition comprises azurin, pseudoazurin, platocyanin, rusticyanin, Laz auracyanin May be part or all of a protein selected from stellacyanin and cucumber basic protein. The pharmaceutical composition may comprise a portion of the peptide selected from SEQ ID NOS: 1-25.

The invention also includes a method. Any method according to the invention can be used to treat a patient suffering from a disease. In certain embodiments of the method according to the invention, the patient is a cancer patient.

Cancers that can be treated in accordance with the present invention include, but are not limited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, rectal cancer, neck, head, bladder, prostate, skin and cervical cancer.

Routes of administration include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patches, suppositories, vitreous injections and oral. In one embodiment, the method comprises a method of treating a patient, wherein the patient is at least 90% identical to (a) a sequence selected from SEQ ID NOS: 26-62 and (b) a sequence selected from SEQ ID NOS: 26-62 Consisting of administering an isolated nucleic acid molecule comprising a polynucleotide sequence selected from the sequence, wherein the isolated nucleic acid sequence is contained in a pharmacologically acceptable carrier and co-administers the cupredoxin peptide.

In another embodiment of the method according to the invention, the method comprises a pharmacologically acceptable carrier to the patient, at least one cupredoxin peptide and (a) a sequence selected from SEQ ID NOS: 26-62 and (b) SEQ ID NOS : At least 90% of the sequence selected in 26-62, comprising administering a pharmaceutical composition comprising an isolated nucleic acid molecule comprising a polynucleotide sequence selected from the same sequence.

In another embodiment of the method according to the invention, the pharmaceutical composition is administered in a manner selected from intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppositories, vitreous injection and oral.

In another embodiment of the method according to the invention, the method comprises a method selected from (a) a sequence selected from SEQ ID NOS: 26-62 and (b) at least 90% identical to a sequence selected from SEQ ID NOS: 26-62 And administering to the patient an isolated nucleic acid molecule comprising a nucleotide sequence, wherein the isolated nucleic acid sequence is contained in a pharmacologically acceptable carrier, co-administers the cupredoxin peptide, and further provides a prophylactic or therapeutic drug. Co-administration.

Cells are also included in the present invention. In one embodiment, the cell has a heterologous gene for azurin derived from Pseudomonas aeruginosa, wherein the cell expresses azurin upon contact with cancer cells. In another embodiment, the coding sequence of azurin in a heterologous gene for azurin is replaced with the coding sequence for the target protein to express the target protein when the cell contacts the cancer cell. In another embodiment, the cell is Pseudomonas aeruginosa and the coding sequence for azurin in the genome is replaced with the coding sequence for the target protein to express the target protein when the cell contacts the cancer cell. In another embodiment, the coding sequence of the azurin in a heterologous gene is replaced with the coding sequence for the target protein such that the cell expresses the target protein upon contact with the cancer cell, and the target protein is a prophylactic protein, therapeutic protein, cytotoxicity. Protein and diagnostic protein.

The invention also includes methods of using the cells described in the paragraphs above. In these specific embodiments, the method consists in treating the patient by administering one or more of the described cell types. The invention also includes a method of diagnosing cancer in a patient using the cell types described in the preceding paragraphs. In one embodiment, the method administers a cell having a heterologous gene for Pseudomonas aeruginosa, wherein the cell expresses azurin upon contact with cancer cells, and the coding sequence of the azurin in the heterologous gene for the target protein. Replaced with a coding sequence, the cell expresses the target protein upon contact with the cancer cell, and the target protein consists of being a diagnostic protein. In other embodiments, the target protein is selected from prophylactic, therapeutic, cytotoxic and diagnostic proteins. In a further embodiment, the pharmaceutical composition is administered in a manner selected from intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection and oral. In this way the patient can treat the patient suffering from the disease and / or cancer. Such features, advantages, and the like of the present invention will become apparent from the following drawings and detailed description.

Brief description of the sequence :

SEQ ID NO: 1 is the amino acid sequence of azurin from Pseudomonas aeruginosa.

SEQ ID NO: 2 is the amino acid sequence of Azurin residues 50-77, p28 from Pseudomonas aeruginosa.

SEQ ID NO: 3 is the amino acid sequence of plastocyanin from Phormidium laminosum .

SEQ ID NO: 4 is Thiobacillus ferrooxidans ).

SEQ ID NO: 5 is Achromobacter Achromobacter cycloclastes ) is an amino acid sequence of pseudoazurin .

SEQ ID NO: 6 is the amino acid sequence of azurin from Alcaligenes faecalis .

SEQ ID NO: 7 is Acromobacter silosooxydans denitrippicans I ( Achromobacter xylosoxidans ssp denitrificans I ) is the amino acid sequence of azurin .

SEQ ID NO: 8 is Bordetella bronchiseptica ) is the amino acid sequence of azurin .

SEQ ID NO: 9 is a Pseudomonas species as mektil (Mcthylomonas sp . ) Is the amino acid sequence of azurin.

SEQ ID NO: 10 is Neisseria meningitides ) is an amino acid sequence of azurin from Z2491.

SEQ ID NO: 11 is Pseudomonas fluorescens ) is the amino acid sequence of azurin .

SEQ ID NO: 12 shows Pseudomonas chlororaphis ) is the amino acid sequence of azurin .

SEQ ID NO: 13 is Xylella fastidiosa ) is the amino acid sequence of azurin from 9a5c.

SEQ ID NO: 14 is the amino acid sequence of Stellacyanin from Cucumis sativus .

SEQ ID NO: 15 is Chloroflexus aurantiacus ) from Auracinin A.

SEQ ID NO: 16 is Chloroflexus aurantiacus ) is the amino acid sequence of auracinin B.

SEQ ID NO: 17 is the amino acid sequence of a cucumber based protein from Cucumis sativus .

SEQ ID NO: 18 is Neisseria meningitides ) is the amino acid sequence of Laz from F62.

SEQ ID NO: 19 is Vibrio enteritis parahaemolyticus ) is the amino acid sequence of azurin .

SEQ ID NO: 20 is Chloroflexus aurantiacus ) from amino acids 57 to 89 of auracinin B.

SEQ ID NO: 21 is the amino acid sequence of amino acids 51-77 of Bordetella pertussis .

SEQ ID NO: 22 is the amino acid sequence of amino acids 89-115 from Neisseria meningitides .

SEQ ID NO: 23 is the amino acid sequence of amino acids 51-77 from Pseudomonas syringae .

SEQ ID NO: 24 is Vibrio (Vibrio parahaemolyticus ). The amino acid sequence of amino acids 52-78.

SEQ ID NO: 25 is Bordetella bronchiseptica ) is the amino acid sequence of amino acids 51-77.

SEQ ID NO: 26 is a 15 kb polynucleotide DNA sequence released from Pseudomonas aeruginosa that encodes a polypeptide similar to Laz of Neisseria .

SEQ ID NOs: 27-62 are 15 kb polynucleotide DNA sequences released from Pseudomonas aeruginosa.

1 shows azurin secretion by Pseudomonas aeruginosa 8821 and 8822 in the presence of cancer cells. 1A shows the growth of two Pseudomonas aeruginosa (8822, 8821) under cancerous cells (MCF-7) or radish (control), and of azurin in extracellular aliquots using Western blotting with anti-azurin antibodies. Release was checked. Panel a: Pseudomonas aeruginosa cell growth shown by measuring turbidity at 600 nm. ■ and ◆ indicate the release levels of azurin with and without cancer cells, respectively. The turbidity difference at 0 min with and without cancer cells is due to the addition of cancer cells. Panel b; Azurin release levels are plotted by signal intensity measurements on Western blotting profiles. ■ and ◆ indicate the release levels of azurin, respectively, in the presence or absence of cancer cells. Panel c: Western blotting. Extracellular aliquots are prepared at 0, 15, 30 and 60 minutes after addition of cancer cells and subjected to SDS-PAGE, Western blotting. The arrow indicates the position of the azurin. 1B. Quantitative Western blotting. Signal intensity in the western blotting profile increases linearly with an increase in standard azurin (0-20 ng). Figure 1C. Azurin production on Pseudomonas aeruginosa 8821 and 8822. The intracellular aliquots obtained during exponential growth of these cells (8821, 8822) were subjected to Western blotting using SDS-PAGE and anti-azurin antibodies. Standard azurin is shown in the left servant. 1D. Azurin was not detected in MCF-7 cell extracts (100 μg protein) by Western blotting.

2 shows a lack of Pseudomonas aeruginosa cell lysis in the presence of cancer cells. Strain 8822 cells transformed with pQF47 plasmid containing the lacZ gene in FIG. 2A were grown with or without cancer cells (MCF-7) or no control (extra), and extracellular and intracellular aliquots were followed by SDS-PAGE followed by anti-lacz Western blotting using the antibody. Sample preparation is the same as in FIG. Standard LacZ is shown in the left line. * Indicates intracellular aliquots of parent strain 8822 cells (without the pQF47 plasmid) incubated with cancer cells for 60 minutes. Figure 2B. Extracellular aliquots of parental strain 8822 cells (transformed with pQF47 plasmid) were Western blotted using SDS-PAGE followed by anti-lacz antibody. 2C shows functional expression of LacZ in 8822 cells (transformed with pQF47 plasmid).

Figure 3 shows in response to the presence of human breast cancer MCF-7 cells the release of cytochrome c ₅₅₁ , but not cytochrome c ₅₅₁ , from the periplasmic between the inner and outer membranes of E. coli cells in an energy dependent manner. Sample preparation is the same as in FIG. Arrows (0 *) indicate intracellular aliquots immediately before culture with cancer. The positions of azurin and cytochrome c ₅₅₁ (cyt c ₅₅₁ ) are indicated by arrows. Figure 3A, azurin secretion from E. coli JM109; Figure 3B Secretion of cytochrome c ₅₅₁ (cyt c ₅₅₁ ) in E. coli JCB7120. 3C, Pseudomonas aeruginosa 8822 is preincubated with 0, 50, 250 μM CCCP for 1 hour to show azurin secretion from these cells. FIG. 3D, E. coli JM109, when pre-incubated with 250 μM CCCP for 1 hour prior to exposure or exposure to MCF-7 breast cancer cells, shows secretion of azurin from these cells. 3E. Effect of CCCP on the Growth of Pseudomonas Aeruginosa 8822 (Left Panel) and Escherichia Coli JM109 (Right Panel). CCCP at the indicated concentration was added at the beginning of the rapid exponential growth phase and continued to grow at the optical density of 600 nm for the next 60 minutes.

Figure 4 shows the release of extrachromosomal DNA of Pseudomonas aeruginosa 8822 strain into the culture medium. 4A, Pseudomonas aeruginosa 8822 strain releases a 15 kb extrachromosomal DNA fragment. DNA was purified from culture medium filtrates by isopropanol precipitation of nucleoprotein aliquots and DNA purification from Qiagen columns (Qiagen, Inc., Valencia CA). 4B shows extrachromosomal DNA secretion at different time points. DNA is released rapidly within 5 minutes and release is enhanced at 15, 30 and 60 minutes, respectively. DNA is effectively released at all times in the presence of MCF-7 human breast cancer cells. 4C, Pseudomonas aeruginosa strain 8822 releases both azurin and 15 kb DNA fragments into the culture medium filtrate in the presence of MCF-7 cancer cells. Strain 8822 was grown in the presence of MCF-7 cancer cells and release of azurin and extrachromosomal DNA from extracellular aliquots was precipitated by PCR and template using primers for Western blotting and PA clones using anti-azurin antibodies Using the prepared DNA. Azurin and DNA release vary over time. 4D. Secreted DNA is CpG rich. DNA is EcoRI. HindIII. Restriction endonuclease enzyme treatment using MspI and PvuI enzymes. MspI and PvuI, known to cleave CpG rich DNA, create an inconsistent DNA band, indicating a high ratio of GC sequences to DNA.

Figure 5 shows the DNA sequence and amino acid translation of very high C and G stretches in released CpG rich DNA. In FIG. 5A such DNA stretch (SEQ ID NOL 49) has no homology with any other sequence in the database and does not appear to be part of an open reading frame. Figure 5B CpG-rich released DNA (8822) (SEQ ID NO : 63) present a very lean virtual genes and Pseudomonas aeruginosa PAO1 and Neisseria gonorrhoeae (Neisseria to the gonorrhoeae ) (NG) (SEQ ID NO: 64) and meningococcal bacterium (NM) (SEQ ID NO: 65) show comparative amino acid sequence homology.

6 shows NF-kB induction by extrachromosomal CpG rich Pseudomonas aeruginosa DNA. 6A, NF-kB induction is TLR9 dependent and requires extrachromosomal CpG-rich DNA. HEK293 cells transformed with TLR9 expression plasmid and NF-kB promoter-induced SEAP (embryonic alkaline phosphatase secretion) reporter plasmid (Invitrogen, Inc. Carlsbad, Calif.) Were treated with various concentrations of CpG-rich 15 kb DNA. SEAP expression after NF-kB activation was measured in supernatants of transfected cells. SEAP levels were quantitatively assessed using the HEK-Blue ™ SEAP Reporter Test Kit (Invitrogen, Inc. Carlsbad, Calif.). Stimulation of HEK293 cells with CpG-rich DNA induces NF-kB to induce SEAP activity in a dose dependent manner. 6B, different cancer cell lines express a wide range of Toll-like receptors (TLRs). TLRs expression was analyzed by quantitative RT-PCR. Data was normalized to the expression of cyclophilin B. Figure 6C. The effect of CpG rich DNA treatment on cell proliferation of MCF-7 breast cancer cells. Cells were treated with various concentrations of 15 kb CpG-rich DNA (0.5 μg, 1 μg, 3 μg, 5 μg) for 12 and 24 hours and cell survival was measured. MTT assays were performed to determine the viability of cells to calculate cell toxicity (rate of cell death), as described previously (Yamada et al, 2002). To calculate the cytotoxicity ratio, the untreated viable cell value was 100% and the number of viable cells treated with 2.5 μg of calf thymus DNA and various concentrations of CpG rich DNA was determined.

7 is a table of CpG rich DNA sequences released from Pseudomonas aeruginosa upon contact with cancer cells.

Justice

As used herein, "cell" encompasses both the singular and the plural of the term unless specifically stated as a "single cell."

In this specification, "polypeptide", "peptide" and "protein" are used as the same meaning, and indicate a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acid. The term also applies to naturally occurring amino acid polymers. "Polypeptides", "peptides" and "proteins" include glycosylation, lipid attachment, sulfation, and gamma-carboxylation and hydroxylation of glutamic acid residues. hydroxylation, ADP-ribosylation, including but not limited to variations. Polypeptides are not always perfectly linear. For example, polypeptides are branched as a result of ubiquitination and generally include post-translational events, including natural processing events and phenomena that do not occur naturally and are caused by human manipulation. as a result of an event) (either branched or unbranched). Cyclic polypeptides, branched polypeptides and branched cyclic polypeptides may be synthesized by non-translated natural processes and entirely by synthetic methods.

As used herein, “polynucleotide”, “oligonucleotide”, “oligo” and “DNA” are used interchangeably to refer to nucleic acid residue polymers.

As used herein, "pharmacological activity" refers to the effect of a drug or other chemical on the biological system. The effects of chemicals may be beneficial (therapeutic) or harmful (toxic). Pure chemicals or mixtures may be of natural origin (plants, animals or minerals) or they may be synthetic compounds.

As used herein, a "pathological condition" refers to an anatomical and physical situation in which a living animal or part of it is damaged from its normal condition and deforms or stops the execution of a body function. It appears in response to various factors (nutrition, industrial pests or climate), infectious agents (worms, parasite protozoa, bacteria or viruses) or genetic combinations of organisms (genetic abnormalities) or combinations thereof.

As used herein, “disorder” encompasses anatomical and physiological deviations from normal, which constitute a steady state injury of a living animal or portion thereof and disrupt or modify the performance of body functions.

As used herein, “suffering” refers to a symptom of the current disorder and encompasses suffering from, recovering from, or recovering from a disorder even though there are no observable symptoms.

As used herein, “treatment” encompasses preventing, decreasing, stopping or reversing the progression or severity of the disorder to be treated or the symptoms associated with the disorder. Thus, "treatment" includes appropriate medical, therapeutic and / or prophylactic administration. Treatment also includes preventing or lessening the occurrence of abnormalities such as cancer.

As used herein, “inhibiting cell growth” means delaying or stopping cell division or cell expansion. The term includes inhibiting cell development or increasing cell death.

A “therapeutically effective amount” is an amount effective to prevent, lower, stop or reverse the occurrence of a particular disorder in the subject being treated, or partially or completely alleviate a particular disorder or existing symptoms of such disorder. Determination of a therapeutically effective amount is within the ability of those skilled in the art.

As used herein, “substantially pure” used to modify a protein or other cell product of the invention is, for example, from a growth medium or cell contents in a form that is substantially free or free of other proteins and / or activity inhibitory compounds. Indicates isolated protein. "Substantially pure" refers to at least 75% factor, or at least "75% substantially pure" factor per dry weight of the separated fractions. More suitably, “substantially pure” refers to at least 85% of the compound per building weight of the active compound, or at least “85% substantially pure” compound. Most suitably, “substantially pure” refers to at least 95% of compounds per building weight of active compound, or at least “95% substantially pure” compounds. “Substantially pure” can also be used to modify a synthesized protein or compound, where, by way of example, such synthetic protein is separated from the reactants and by-products of the synthetic reaction.

As used herein, a “pharmaceutical grade” in connection with a peptide or protein of the present invention is substantially or essentially separated from components commonly associated in nature, including synthetic reactants and by-products, and as pharmaceuticals thereof. It indicates a peptide or compound that is substantially or essentially separated from the component that constrains its utilization. For example, “pharmaceutical grade” peptides are isolated from any carcinogen. In some instances, “pharmaceutical grade” is modified by the intended method of administration to specify a peptide or compound that is substantially or essentially separated from any material that makes the composition unsuitable for intravenous administration to a patient ( Eg, “intravenous pharmaceutical grade”). For example, “intravenous pharmaceutical grade” peptides are isolated from detergents such as SDS and anti-bacterial agents such as azide.

“Isolated”, “purified” or “biologically pure” refers to a substance that is substantially or essentially free of components normally involved in its natural state. Thus, isolated peptides according to the invention are preferably in situ It does not include substances commonly associated with these peptides in the environment. "Isolated" region refers to a region in which such region does not carry the entire sequence of the polypeptide from which it is derived. “Isolated” nucleic acids, proteins, or individual fragments thereof may be subjected to nucleotide sequencing, restriction digestion, site-directed mutagenesis, or subcloning into expression vectors for nucleic acid fragments. subcloning) and acquisition of proteins or protein fragments in substantially pure amounts, substantially transferred from the in vivo environment by manipulation by those skilled in the art, including, but not limited to.

As used herein, “variant” refers to an amino acid sequence variant that has been substituted, deleted, or inserted into an amino acid, as compared to a wild type polypeptide. The variant may be truncation of the wild type peptide. "Deletion" is the removal of one or more amino acids in a wild-type protein and "fragment" is the removal of one or more amino acids from one or more ends of a wild-type protein. Thus, variant peptides are made by manipulation of genes encoding such polypeptides. Variants are made by changing the basic composition or properties without changing at least some of the fundamental pharmacological activity of the polypeptide. For example, the "variants" of azurin can be mutated azurins that retain their ability to inhibit the development of premalignant mammalian cells. In some instances, variant peptides are synthesized with non-natural amino acids such as ε- (3,5-dinitrobenzoyl) -Lys residues (Ghadiri & Fernholz, J. Am. Chem. Soc., 112: 9633). -9635 (1990)). In some embodiments, a variant is substituted, deleted or inserted up to 20 amino acids as compared to a wild type peptide or portion thereof. In some embodiments, the variant has no more than 15 amino acids substituted, deleted or inserted compared to the wild type peptide or a portion thereof. In some embodiments, the variant is substituted, deleted or inserted with up to 10 amino acids as compared to the wild type peptide or portion thereof. In some embodiments, a variant is substituted, deleted or inserted with up to 6 amino acids as compared to a wild type peptide or portion thereof. In some embodiments, a variant is substituted, deleted or inserted with up to 5 amino acids as compared to a wild type peptide or portion thereof. In some embodiments, a variant is substituted, deleted or inserted with up to 3 amino acids as compared to a wild type peptide or portion thereof.

As used herein, an “amino acid” is an amino acid moiety comprising any naturally-occurring or non-naturally occurring or synthetic amino acid residue, ie one, two, three or more carbon atoms, typically By any moiety comprising at least one carboxyl residue and at least one amino residue directly linked by one (α) carbon atom.

As used herein, “derivative” refers to a peptide derived from a target peptide. Derivation includes chemical modifications of the peptide such that the peptide still retains some of its underlying pharmacological activity. For example, the "derivative" of azurin may be a chemically modified azurin that maintains its ability to inhibit angiogenesis in mammalian cells. Desired chemical modifications include, but are not limited to, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, glycosylation of peptides. In addition, the derivative peptide may be a fusion of a polypeptide to a chemical compound, eg, another peptide, a drug molecule or other therapeutic or pharmaceutical agent, or a detectable probe.

As used herein, “% amino acid sequence identity” is defined as the ratio of amino acid residues in a polypeptide that matches amino acid residues in a candidate sequence when the two sequences are aligned. To determine the percent amino acid identity, the sequences are aligned and, if necessary, a gap is introduced to ensure the maximum percent sequence identity; Conservative substitutions are not considered as part of sequence identity. Amino acid sequence alignment procedures to determine percent identity are known to those skilled in the art. Publicly available computer software, such as BLAST, BLAST2, ALIGN or Megalign (DNASTAR) software, is used to align peptide sequences. In certain embodiments, Blastn (available from the National Center for Biotechnology Information, Bethesda MD) is used in a default parameter of low complexity filter, expect 10, word size 11.

"Host cells" include individual cells or cell cultures that have been, or are likely to be, receptors of isolated polynucleotides or any recombinant vectors of the invention. and naturally, it is not highly required of the completely the same due to the mutation and or change due to an accident (in the form or the entire DNA complement), the host cell a recombinant vector or a polynucleotide of the present invention and in vivo or in Includes infected or infected cells in vitro .

“Transformation” has the same meaning as “genetic change”, which refers to a permanent or transient genetic modification induced in a cell after the introduction of new DNA (eg, exogenous to the cell). Genetic changes (“modifications”) are made by binding new DNA into the genome of a host cell or by the routine or stable maintenance of new DNA as an episomal element.

When aligning the amino acid sequence, the amino acid sequence identity ratio of the constant amino acid sequence A to the constant amino acid sequence B (or the constant amino acid sequence A having or including the specific% amino acid sequence identity to the constant amino acid sequence B) (%) Can be calculated as follows:

% Amino acid sequence identity = X / Y * 100

Where X is the number of amino acid residues that are recorded in the same match of A and B by sequence alignment program or algorithm alignment,

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percentage amino acid sequence identity of A to B will not match the percentage amino acid sequence identity of B to A. When comparing longer sequences to shorter sequences, the shorter sequence will be the “B” sequence. For example, when comparing polypeptides truncated to the corresponding wild type polypeptide, the truncated peptide will be the “B” sequence.

In aligning nucleotide sequences, the ratio of nucleotide sequence identity of a constant nucleotide sequence A to a constant nucleotide sequence B (or a constant nucleotide sequence A having or including a specific percentage of nucleotide sequence to a constant nucleotide sequence B) (%) Can be calculated as follows:

% Nucleotide Sequence Identity = X / Y * 100

Where X is the number of nucleotide residues that are recorded in the same match of A and B by sequence alignment program or algorithm alignment,

Y is the total number of nucleotide residues in B.

If the length of nucleotide sequence A is not equal to the length of nucleotide sequence B, the percentage nucleotide sequence identity of A to B will not match the percentage nucleotide sequence identity of B to A. When comparing longer sequences to shorter sequences, the shorter sequence will be the “B” sequence. For example, when comparing polypeptides truncated to the corresponding wild type polypeptide, the truncated peptide will be the “B” sequence.

General

The present invention provides a composition composed of isolated polynucleotides comprising CpG rich DNA derived from Pseudomonas aeruginosa. The present invention also provides a pharmaceutical composition comprising CpG rich DNA derived from Pseudomonas aeruginosa and optionally at least one cupredoxin peptide, which treats a patient, in particular a patient suffering from a disorder, in particular a patient suffering from cancer. It will be beneficial to prevent this. The present invention also provides a method for treating a patient, in particular for treating a patient suffering from a disorder, in particular a cancer, or preventing cancer, which method comprises CpG rich DNA derived from Pseudomonas aeruginosa, optionally in combination with a cupredoxin peptide. It consists of. The present invention also provides cells which induce expression of specific proteins upon contact with cancer cells using azurin derived from Pseudomonas aeruginosa. These cells can be used in the treatment of patients, in particular in the treatment of patients suffering from disorders, in particular in cancer, or in the prevention of cancer or in the diagnosis of cancer in patients.

In connection with the first aspect of the present invention, it is already known that cupredoxin azurin, a redox protein caused by Pseudomonas aeruginosa, selectively invades J774 lung cancer cells rather than normal cells to induce apoptosis. Zaborina et al, Microbiology 146: 2521-2530 (2000). Azurin can selectively invade human melanoma cells UISO-Mel-2 or human breast cancer MCF-7 cells. Yamada et al. PNAS 99: 14098-14303 (2002); Punj et al., Oncogene 23: 2367-2378 (2004). Azurin of Pseudomonas aeruginosa preferentially invades J774 murine retinal cell sarcoma cells, complexes with and stabilizes the tumor suppressor protein p53, increasing the intracellular concentration of p53 and inducing apoptosis. Yamada et al. Infection and Immunity 70: 7054-7062 (2002)

In studies of the various domains of the azurin molecule, azurin 50-77 (p28) (SEQ ID NO: 2) exhibits a protein conversion domain (PTD), which is important for apoptotic activity in refractory sequence. Yamada et al. Cell. Microbial. 7: MIS-MS 1 (2005). Pseudomonas aeruginosa is also known to release specific DNA sequences in a manner that is enhanced in the presence of cancer cells with extracellular media. See Example 5. Such DNA is released from Pseudomonas aeruginosa within 5 minutes after contact with cancer cells, suggesting that the released DNA is originally extrachromosomal DNA. See Example 6. When the released DNA is treated with restriction enzymes, it is called "CpG-rich DNA" because the DNA contains many G + C nucleotides. See Example 7. Of the DNA sequences found among the released sequences, the SEQ ID NO: 26 sequence is CpG rich and is 95% homologous to the nucleotide sequence of lax, the azurin gene of Neisseria . See Example 8. Finally, such Pseudomonas aeruginosa CpG rich DNA preparations are currently known to have anti-tumor properties, which is explained by their ability to activate NF-kB in a TLR9 dependent manner.

Administration of Pseudomonas aeruginosa CpG rich DNA with cupredoxin is expected to prevent or treat cancer in patients, particularly those suffering from disorders, especially those suffering from cancer. In particular, polynucleotides released from Pseudomonas aeruginosa having a sequence very similar to the lax gene of Neisseria , SEQ ID NO: 26, may be coadministered with cupredoxin, such as the azurin of the Pseudomonas aeruginosa or the 50-77 residue portion (p28) of azurin. In the treatment of cancer, AIDS, malaria, inadequate angiogenesis, or the risk of developing cancer, the effect may be improved when compared to cupredoxin alone. Although not limited to any method of treatment, CpG rich DNA released by Pseudomonas aeruginosa is considered to reduce the extent to which the patient's immune system attacks co-administered cupredoxin peptides. Pseudomonas aeruginosa developed as a parasite in mammalian species, and tumor growth in mammalian species inhibits Pseudomonas aeruginosa growth. Pseudomonas aeruginosa is a weapon against cancer and actively releases azurin upon exposure to cancer cells. Because azurin can be a target of the host immune system for antibody formation, Pseudomonas aeruginosa strain 8822 can be used as a handout to disrupt the immune system that targets azurin by releasing CpG rich DNA that is released upon exposure to cancer cells. . Since CpG rich DNA appears to protect selectively co-administered cupredoxin, it will be effective in improving the effectiveness of any treatment method of administering cupredoxin regardless of the disease or disorder being treated. CgG rich DNA is also considered to be useful for protecting any co-administered chemical targeted by the immune system.

For a second aspect of the invention, the azurin of Pseudomonas aeruginosa is a periplasmic protein that is released into the growth medium at later stages of growth. Zaborina et al., Microbiology 146: 2521-2530 (2000). This extracellular release relies on quorum sensing at high cell densities and is regulated by GacA or two small RNA products. RsmY and RsmZ. Kay et al., J. Bacterid. 188: 6026- 6033 (2006).

It is also now known that the presence of human cancer cells induces Pseudomonas aeruginosa to release azurin into the extracellular medium. In the presence of cancer cell lines MCF-7 and Mel-2, Pseudomonas aeruginosa releases azurin into the medium within 20-30 minutes of exposure to cancer cells, but no cancer cells were observed to be free. See Example 1. In addition, such azurin release is not due to cell lysis but requires the substantial presence of cancer cells in contrast to the proliferative factors of cancer cells. See Example 2. It is also known that cancer cells will induce the release of azurin from E. coli carrying the Pseudomonas aeruginosa azurin gene. See Example 3. Finally, no energy is required for azurin release from Pseudomonas aeruginosa or E. coli. See Example 4.

The living cells of M. bovis are widely used in the treatment of superficial bladder cancer, and the use of attenuated bacteria in potential treatments for cancer attracts considerable attention. Chakrabarty, J. Bacterial. 185: 2683-2686 (2003); Minton, Nature Rev. Microbiol 1: 237-243 (2003); Dang et al, Cancer Biol. Therap., 3: 326-33? (2004): Vassaux et al., I Pathol. 208: 290-298 (2006). The azurin gene of Pseudomonas aeruginosa is considered to be used in Pseudomonas aeruginosa or other cells such as E. coli to express azurin or dysplastic proteins upon contact with cancer cells. The useful use of cells with an azurin gene or a gene expressing a heterozygous protein may provide a means for carrying a protein expressed at a cancer cell site to treat cancer or to diagnose cancer cell location by protein expression. Could be

Compositions of the Invention

The present invention provides isolated polynucleotides that are released from Pseudomonas aeruginosa and that would be useful for treating patients, particularly those suffering from disorders, particularly cancer. In some embodiments, the isolated polynucleotide is CpG rich DNA. In some embodiments, the isolated polynucleotide is released from Pseudomonas aeruginosa upon contact with cancer cells and is prepared according to the procedures provided in Example 5. In other embodiments, the isolated CpG rich DNA is one or more isolated polynucleotides having the sequence provided in SEQ ID NOS: 26-62. In certain embodiments, the isolated CpG rich DNA is an isolated polynucleotide having the sequence of SEQ ID NO: 26. In another specific embodiment, the CpG rich DNA consists of isolated polynucleotides having the sequences of SEQ ID NOS: 26-62.

The present invention provides a composition comprising isolated CpG rich DNA released from Pseudomonas aeruginosa and optionally at least one isolated cupredoxin peptide. In some embodiments, such compositions include a pharmacologically acceptable carrier. In certain embodiments, the compositions are designed to, but are not limited to, routes of administration of oral, peritoneal, or intravenous. Such compositions may be hydrated in water or may be dried (frozen dried) for later hydration. Such compositions may be included in solvents other than water, such as alcohols.

CpG rich DNA may be in the form of stabilized DNA. In one embodiment, the DNA is treated with calcium chloride in 20% (v / v) tert-butanol to make the DNA particles of DNA (around 50 nm) as well as larger (about 300 nm) rods or spheres. Can be stabilized by condensation. Condensed particles have a negative surface charge, which indicates a sub-stoichiometric concentration of calcium. More specifically, in one embodiment, purified deionized DNA in a concentration range of about 0.1 μg / ml to about 1 mg / ml contains an aqueous solution of t-butanol in a range of about 17% to about 25% (v / v). Can be dissolved in. Then Ca ^{2 +,} Mg ^{2 +} or suitable divalent containing Zn ^{+ 2} was added to t- butanol co-solvent for the cation of about 0.2nM to about 2nM concentration range thereby condense the DNA. In one embodiment, the stoichiometric ratio of the anionic phosphate and dicationic of the DNA backbone is about 0.1 to about 1.0 (anion / cationic) and about 0.3 is the most suitable ratio. The solution is then equalized for about 45 minutes to create thermodynamic equilibrium condensation. The rod, rotator and spherical DNA particles range in size from about 20 nm to about 500 nm. Solutions containing condensed DNA are used in downstream process unit operations, including but not limited to sterile filtration, spray-drying or lyophilization, such as passing through a 0.22 μm filter.

Sterile filtered condensed DNA is lyophilized or spray dried to obtain a stable pharmaceutical unit form. Prior to lyophilization, the DNA may be complexed with sucrose, mannitol, trehalose or other conventional bulking agents. The t-butanol solution is frozen and forms a single phase that can be lyophilized due to sublimation of t-butanol. In the dried lyophilized cake, the DNA rotator and rods remain intact. Upon reconstitution, the lyophilized cake quickly dissolves and the DNA is completely dissolved to form uncondensed native plasmid DNA with a positive amount of cation and any added bulking agent inherent in the process. Substantially free t-butanol in this step of the process.

Another way to process DNA is to use spray drying. Spray drying can involve three basic unit processes; Liquid atomization, gas-droplet mixing and drying from liquid droplets. Atomization is typically performed by one of three atomization devices (high pressure nozzles, two fluid nozzles, and high pressure centrifugal discs). With these nebulizers, thin solutions will be dispersed into small droplets, such as about 2 μm. The maximum drop size rarely exceeds about 500 μm (35 mesh). Due to the large overall dry surface and the small droplet size created, the actual drying time in the spray dryer generally does not exceed about 30 seconds.

One of the basic advantages of spray drying is to make spherical particles, which are not normally obtainable by other drying methods. The spherical particles may be solid or pupil, depending on the material, feed conditions and drying conditions. The high heat-transfer rate to the drop causes the liquid to evaporate in the center of the particle, causing the outer shell to expand, creating a pupil sphere.

The dried DNA particles can be reconstituted directly in a hydration solution for extra-intestinal administration, including but not limited to muscle and peritoneal administration, using powdered DNA forms. The reconstituted DNA may be administered subcutaneously or ocularly. The reconstituted DNA may be administered via aerosol means or may be delivered as a dry granulated powder by aerosol or other inhalation means. The powdered DNA compositions for the formulations are substantially free of solvents used to substantially modify these structures.

The isolated polynucleotide may be similar to, but not identical to, one or more DNA sequences found in the DNA released by Pseudomonas aeruginosa upon contact with cancer cells. In one embodiment, an isolated polynucleotide may have at least about 80% nucleotide sequence homology with one or more of SEQ ID NOS: 26-62. In another embodiment, an isolated polynucleotide may have at least about 90% nucleotide sequence homology with one or more of SEQ ID NOS: 26-62. In another embodiment, an isolated polynucleotide can have at least about 95% nucleotide sequence homology with one or more of SEQ ID NOS: 26-62.

The composition of the present invention may further comprise a cupredoxin peptide having other pharmacological or antitumor activity of interest.

Cupredoxin peptides can be cupredoxin, variants thereof, derivatives thereof and structural equivalents thereof, such as U.S. Patent Application Serial No. 10 / 047,710, Jan. 15, 2002 (U.S. Patent Serial No. 7,084,105); U.S. Patent Application Serial No. 10,720,603, Nov. 11, 2003; U.S. Patent Application Serial No. 11 / 244,105, October 6, 2005, U.S. Patent Application Serial No. 11 / 488,695, July 19, 2006; U.S. Patent Application Serial No. 11 / 436,592, May 19, 2006; U.S. Patent Application Serial No. 11 / 488,693, July 19, 2006; U.S. Patent Application Serial No. 11 / 436,590 May 19, 2006; U.S. Patent Application Serial No. 11 / 436,591, May 19, 2006; U.S. Provisional Patent Application Serial No. 60/843, 388, September 11. 2006; U.S. Provisional Patent Application Serial No. 60 / 844,358, September 14, 2006, each of which is incorporated by reference. In one embodiment, the cupredoxin peptide may have at least one pharmacological activity of cupredoxin. Certain pharmacological activities of interest include (1) ability to invade mammalian cancer cells, (2) ability to invade non-cancerous mammalian cells, (3) ability to invade pre-malignant mammalian cells, and (4) mammalian cancer. Apoptosis ability, (5) pre-malignant mammalian cell killing ability, (6) mammalian cancer cell killing ability, (7) HIV-1 infection inhibition ability, (8) malaria-infected red blood cells, parasitic inhibition ability, ( 9) epine signaling system interference ability, (10) angiogenesis inhibitory ability, and (11) inhibition of precancerous lesion development.

The cupredoxin peptide may be a full length wild type cupredoxin or a variant, derivative or functional equivalent thereof that exhibits one or more pharmacological activity in mammalian cells, tissues or animals. In some embodiments, the cupredoxin peptide is isolated or in some embodiments, the cupredoxin peptide is of substantially pure or pharmacological grade. In other embodiments, the cupredoxin peptide may comprise or consist of essential peptides. In certain other embodiments, the cupredoxin does not elicit an immune response in mammals, particularly humans. In some embodiments, the cupredoxin peptide is smaller than the full length cupredoxin and has only a portion of the pharmacological activity of cupredoxin.

Because of the high structural homology between cupredoxins, cupredoxin peptides are considered to have the same pharmacological activity as p28 (SEQ ID NO: 2). In some embodiments, the cupredoxin peptide is or is not limited to azurin, pseudoazurin, plastocyanin, rustiyanin, Laz or auracyanin. In certain embodiments, the azurin is Pseudomonas aeruginosa, Alkagen's Pecalis, Acromobacter silosooxydans. Dennitry Parkans ( Achromobacter) xylosoxidans ssp . denitrificans I ), Bordetella bronchiceptica, Mectillomonas spp., meningitis, gonococcus, Pseudomonas fluescins, Pseudomonas chlorolapis, cilera pastiosa, enteritis vibrio. In a very specific embodiment, azurin is derived from Pseudomonas aeruginosa. In another specific embodiment, the cupredoxin peptide comprises an amino acid sequence which is the sequence of SEQ ID NOs: 1, 3-19.

Cupredoxin derived peptides are amino acid sequence variants that are substituted, deleted, or inserted amino acids in comparison to wild type cupredoxin. These variants may be truncated of wild type cupredoxin. Cupredoxin derived peptides comprise a region of cupredoxin, which is a wild-type polypeptide of up to full length. In some embodiments, the cupredoxin derived peptide comprises at least 10 residues, at least 15 residues, or at least 20 residues of truncated cupredoxin. In some embodiments, the cupredoxin derived peptide comprises up to 100 residues, up to 50 residues, up to 40 residues, up to 30 residues or up to 20 residues of truncated cupredoxin . In some embodiments, the cupredoxin is a cupredoxin derived peptide, more specifically, at least 70% amino acid sequence identity, at least 80% amino acid sequence identity, at least 90% amino acid sequence identity, at least in SEQ ID NOs: 1-19 95% amino acid sequence identity or at least 99% amino acid sequence identity.

In certain embodiments, the cupredoxin derived variant comprises Pseudomonas aeruginosa azurin residues 50-77, azurin residues 50-67, or azurin residues 36-88. In another embodiment, the variant of cupredoxin consists of Pseudomonas aeruginosa azurin residues 50-77, azurin residues 50-67, or azurin residues 36-88. Other cupredoxin peptides having similar activity to Pseudomonas aeruginosa azurin residues 50-77, azurin residues 50-67, or azurin residues 36-88 may be designed. To this end, the target cupredoxin amino acid sequence is arranged in the Pseudomonas aeruginosa sequence using BLAST, BLAST2, ALIGN2, or Megalign (DNASTAR), whereby Pseudomonas aeruginosa (the related residue located in the azurin amino acid sequence and the equivalent found in the target cupredoxin sequence Aligned to residues, and therefore, equivalent peptides are designed.

In one embodiment of the invention, the cupredoxin-derived peptide is Chloroflexus aurantiacus ) (SEQ ID NO: 20) at least amino acids 57 to 89 of auracyanin B. In another embodiment of the invention, the cupredoxin derived peptide comprises at least amino acids 51-77 (SEQ ID NO: 21) of Bordetella pertussis azurin. In another embodiment of the invention, the cupredoxin variant comprises at least amino acids 89-115 of Pseudomonas syringae (SEQ ID NO: 23). In another embodiment of the invention, the cupredoxin variant is Neisseria meningitidis ) at least amino acids 89-115 of Laz (SEQ ID NO: 22). In another embodiment of the invention, the cupredoxin derived peptide is Vibrio parahaemolyticus ) contains at least amino acids 52-78 (SEQ ID NO: 24) of azurin. In another embodiment of the invention, a single pre-ku derived peptides Bordetella chevron kisep urticae (Bordetella bronchiseptica ) contains at least amino acids 51-77 (SEQ ID NO: 29) of azurin.

Cupredoxin derived peptides also include peptides made from synthetic amino acids that do not occur naturally. For example, non-naturally occurring amino acids are incorporated into variant peptides to extend or optimize the half-life of the composition in the bloodstream. Such variants include D, L-peptides (diastereomers) (eg, Futaki et. al ., J. Biol. Chem. 276 (8): 5836-40 (2001); Papo et al . Cancer Res. 64 (16): 5779-86 (2004); Miller et al , Biochem. Pharmacol. 36 (1): 169-76, (1987)), peptides with special amino acids (eg, Lee et al ., J. Pept. Res. 63 (2): 69-84 (2004)), olefin-bearing non-natural amino acids followed by hydrocarbon stapling (eg, Schafmeister et. al . , J. Am. Chem. Soc. 122: 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)), peptides comprising ε- (3,5-dinitrobenzoyl) -Lys residues are included, but are not limited to these.

In another embodiment, the cupredoxin derived peptide is a derivative of cupredoxin. Derivatives of cupredoxin are chemical modifications of the peptide such that the peptide still retains some of its underlying pharmacological activity. For example, the "derivative" of azurin can be a chemically modified azurin that maintains its ability to inhibit the growth of mammalian cancer cells. Desired chemical modifications include, but are not limited to, hydrocarbon stapling, amidation, acetylation, sulfation, polyethylene glycol (PEG) modification, phosphorylation, glycosylation of peptides. It doesn't work. In addition, the derivative peptide may be a fusion of a cupredoxin, or variant, derivative or structural equivalent thereof to a chemical compound, such as another peptide, pharmaceutical molecule or other therapeutic or pharmaceutical agent, or detectable probe. Derivatives of interest include, for example, circularized peptides (eg, Monk et al . , BioDrugs 19 (4): 261-78, (2005); DeFreest et. al ., J. Pept. Res. 63 (5): 409-19 (2004)), N- and C-terminal modifications (eg, Labrie et. al ., Clin. Invest. Med. 13 (5): 275-8, (1990)), incorporation of olefin-bearing non-natural amino acids followed by hydrocarbon stapling (eg, Schafmeister et al . , J. Am. Chem. Soc. 122 : 5891-5892 (2000); Walenski et al ., Science 305: 1466-1470 (2004)), including but not limited to, chemicals that can extend or optimize the half-life of the peptides and compositions of the invention in the bloodstream by a number of methods well known to those skilled in the art. Hold the variant.

In addition, the cupredoxin peptide may be a form in which a cupredoxin peptide is fused to a cargo such as a chemical compound, which includes other peptides, drug molecules or therapeutic or pharmaceutical substances, or detectable probes. It is not limited to this. In one embodiment, the detectable substance is such that, for example, a substance such as a phlororesin protein, a luminescent substance, an enzyme such as beta-galactoside or a radiolabeled or biotinylated protein imparts a detectable phenotype to the cell. To be transported. Similarly, microparticles or nanoparticles labeled with a material such as fluorescent material may be transported. Suitable nanoparticles are described in U.S. Pat. No. 6,383,500, (registered May 7, 2002, attached as a reference). Many such detectable materials are known in the art.

In some embodiments, the cargo compound is a detectable material suitable for X-ray tomography, magnetic resonance imaging, ultrasound imaging or radionuclide imaging. In such embodiments, the cargo compound is administered to the patient for diagnostic purposes. Contrast agents are administered as cargo compounds to enhance the images obtained by X-ray CT, MRI and ultrasound. Administration of a nuclide cargo compound targeted to tumor tissue through the cupredoxin entry domain can be used for nuclide imaging. In some embodiments, the cupredoxin invasion domain may include a nuclide with or without a cargo compound. In other embodiments, the compound can be a gamma ray or positron emitting isotope, a magnetic resonance imaging contrast agent, an X-ray contrast agent, or an ultrasound contrast agent.

Ultrasonic contrast agents suitable for use as cargo compounds include, but are not limited to, selective linking moieties between target moieties and microbubbles, microbubbles of biocompatible gases including Ln, liquid carriers, and surfactant microspheres. In the present context, a liquid carrier includes an aqueous solution, and a surfactant means any amphoteric material that reduces the interfacial tension of the solution. A list of suitable surfactants that form surfactant microspheres can be found in EP0727225A2, which is hereby incorporated by reference. Surfactant microspheres include nanospheres, liposomes, vesicles and the like. The biocompatible gas can also be an atmosphere, a hydrocarbon, such as C ₃ -C ₅ perfluoroalkane (provides a difference in the absorption factor (echogenicity) against the ultrasound image to make the difference). The gas is trapped or contained in microspheres and attaches to the cupredoxin invasion domain via a linker. Attachment can be covalent, ionic or van der Waals forces. Specific examples of such contrast agents include perfluorocarbons trapped in lipids having a number of tumor neovascular receptor receptor peptides, polypeptides, and peptidomimetic.

X-ray contrast agents suitable for use as cargo compounds include one or more X-ray absorption or “heavy” atoms of 20 or more atoms, optionally optionally between the cupredoxin invasion domain and the X-ray absorbing atoms. Linking moiety, Ln. The heavy atom commonly used in X-ray contrast agents is iodine. Recently, X-ray contrast agents have been disclosed that include metal chelates (eg US Pat. No. 5,417,959) and polychelates comprising a plurality of metal ions (eg US Pat. No. 5.679,810). More recently, multinuclear cluster complexes are known as X-ray contrast agents (eg, U. S. Pat. No. 5,804, 161, PCT WO 91/14460, PCT WO 92/17215).

MRI contrast agents suitable for use as cargo compounds include, but are not limited to, one or more paramagnetic metal ions and, optionally, a linking moiety, Ln, between the cupredoxin invasion domain and the X-ray absorbing atom. Paramagnetic metal ions exist in the form of metal complexes or metal oxide particles. U.S. Pat. Nos. 5,412,148, and 5,760,191 describe examples of paramagnetic metal ion chelates that can be used as MRI contrast agents. U.S. Pat. No 5,801,228. U.S. Pat. No. 5.567,41 1, U.S. Pat. No. 5,281,704 describes examples of polychelating agents useful in one or more paramagnetic metal ion complexes that can be used as MRI contrast agents. U.S. Pat. No. 5,520,904 describes granular compounds composed of paramagnetic metal ions that can be used as MRI contrast agents.

In another embodiment, the cargo compound is delivered to slow or kill cell cycle progression in cells such as cancer cells. Such cancer cells can be, for example, osteosarcoma cells, lung carcinoma cells, colon carcinoma cells, lymphoma cells, leukemia cells, soft tissue sarcoma cells, breast, liver, bladder, or prostate carcinoma cells. For example, the cargo compound may be a cell cycle regulatory protein such as p53; Cyclin-dependent kinase inhibitors such as p16, p21 or p27; Suicide proteins such as thymidine kinase or nitroreductase; Cytokines or other immunomodulatory proteins such as interleukin 1, interleukin 2 or granulocyte-macrophage colony stimulating factor (GM-CSF); Or a toxin such as Pseudomonas aeruginosa exotoxin A. In another embodiment, the bioactive fragment of one of the compounds is delivered.

In another embodiment, the cargo compound is a nucleic acid encoding one of the compounds. In another embodiment, the cargo compound is a drug used to treat cancer. Such drugs include 5-fluorouracil, interferon alpha; Methotrexate, tamoxifen; Vincristine and the like. The above examples are for illustrative purposes and many optional other compounds are known in the art.

Cargo compounds suitable for treating cancer include alkylating substances such as nitrogen mustard, alkyl sulfonates, nitrozoreas, ethyleneimine and triazene; Anti-metabolites such as hydrochloric acid antagonists, purine analogs, pyrimidine analogs; Antibiotics such as anthracyclines, bleomycin, mitomycin, dactinomycin and plicamycin; Enzymes such as L-asparaginase; Farnesyl-protein transferase inhibitors; 5. alpha-reductase inhibitors; 17β-hydroxysteroid dehydrogenase type 3; Hormonal substances such as glucocorticoids, estrogens / antiestrogens, androgens / antiandrogens, progestins and progesterone-releasing hormone antagonists, octreotide acetates; Microtube-rupturing materials such as ecteinascidin or analogs and derivatives thereof; Microtube-stabilizing materials such as taxanes such as paclitaxel, docetaxel and the like and epothilones such as epotyrone A-F and the like; Plant-derived products such as vinca alkaloids, epipodophyllotoxins, taxanes; And topoisomerase inhibitors; Prenyl-protein transferase inhibitors; And other substances such as hydroxyurea, procarbazine, mitotan, hexamethylmelamine, platinum coordination complexes such as cisplatin and carboplatin; And other substances used as anticancer substances and cytotoxic substances such as biological response modifiers and growth factors; Immune modulators and monoclonal antibodies, and the like.

Representative examples of anticancer and cytotoxic substances include mechloretamine hydrochloride, cyclophosphamide, chlorambutyl, melparan, ephosphide, busulfan, carmustine, lomustine, semustine, streptozosin, thiotepa, Dacarbazine, methotrexate, thiogamine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine, prourouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mito Mycin C, actinomycin D, saprasin, sapramycin, quinocarcin, discodermoride, vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate, teniposide, Paclitaxel, tamoxifen, esturamustine, esturamustine phosphate sodium, flutamide, busererin, leuprolide, pteridines, levamisol, aflacons, Interferon, enterokin, aldesrukin, philgrastim, sargramostin, rutushimab, BCG, tretinoin, irinotecan hydrochloride, betamethosone, gemcitabine hydrochloride, altretamine, topoteca and analogs thereof or Derivatives thereof and the like, but is not limited thereto.

Preferred among these types are paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, metopethene C, exedenasidine 743, or porphyromycin, 5-fluoro Uracil, 6-mercaptopurine, gemcitabulin, cytosine arabinoside, grapephytotoxin or grapephytotoxin derivatives such as etoposide, etoposide phosphate or teniforce, melphalan, vinblastine, Vincristine, luroshidine, vindesine and lurosine, and the like.

Examples of anticancer and other cytotoxic substances useful as cargo compounds include: Epotyrone derivatives, German Patent No. 4138042.8; WO 97/19086, WO 98/22461. WO 98/25929, WO 9838192, WO 99/01124. WO 99/02224, WO 99O2514. WO 99/03848, WO 99/07692, WO 99 27890, WO 99 28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/65913, W7O 99/67252, WO 99 / 67253, WO 00 / O0485; Cyclin dependent kinase inhibitors WO 99/24416 (U.S. Pat. No. 6.040.321): prenyl-protein transferase inhibitors WO 97/30992 and WO 98/54906; U.S. Pat, No. Substances described in 6,011,029 (compounds in the US patent can be used with other NHR modulators, which include AR modulators, ER modulators, LHRH modulators or surgical castrations, especially modulators used in cancer treatment). Including but not limited to).

Other therapeutic agents that can be used as cargo compounds with the compounds of the present invention can be used in Physicians' Desk Reference (PDR) or in amounts known in the art.

In another embodiment, the cupredoxin peptide is a structural equivalent of cupredoxin. Studies that determine significant structural similarities between cupredoxin and other proteins include those of Toth et al, (Developmental Cell 1: 82-92 (2001)). In particular, significant structural similarities between cupredoxin and structural equivalents are determined using the VAST algorithm. Gibrat et al, Curr Opin Struct Biol 6: 377-385 (1996); Madej et al, Proteins 23: 356-3690 (1995). In certain embodiments, the VAST p value in the structural comparison of cupredoxin to cupredoxin peptide is less than 10 ^−3, less than 10 ⁻⁵ , or less than 10 ⁻⁷ . In another embodiment, the significant structural similarity of the cupredoxin with the structural equivalent is determined using the DALI algorithm. Holm & Sander, J. MoI. Biol. 233: 123-138 (1993). In certain embodiments, the DALI Z score for pairwise structural comparison is at least about 3.5, at least about 7.0, or at least about 10.

Cupredoxin peptides are contemplated to be one or more variants, derivatives or functional equivalents of cupredoxin. For example, the cupredoxin peptide can become the truncated form of pegylated azurin to make both variants and derivatives. In one embodiment, the cupredoxin peptide is synthesized with tetherα, α-disubstituted non-natural amino acids with olefins and then “staples” all hydrocarbons by ruthenium catalyzed olefin metathesis. Scharmeister et al, J. Am. Chem. Soc. 122: 5891-5892 (2000); Walensky et al. Science 305: 1466-1470 (2004) .Cupredoxin peptides, which are structural equivalents of azurin, are additionally peptides. Peptides that are structural equivalents and derivatives can be fused to to merely illustrate, but are not limited to, the present invention The cupredoxin peptide may or may not bind copper.

In some embodiments, the cupredoxin peptide retains some of the functional characteristics of Pseudomonas aeruginosa azurin, in particular p28. In certain embodiments, the cupredoxin peptide may also inhibit angiogenesis in mammalian cells, tissues or animals, particularly including but not limited to HUVECs. Cupredoxin peptides may also possess the ability to inhibit the growth of mammalian cancer cells, including but not limited to melanoma, breast, pancreas, glioma, astrocytoma or lung cancer cells. Cupredoxin peptides may also possess the ability to inhibit the development of adenocarcinoma mammalian cells. Cupredoxin peptides possess the ability to invade cancer cells, including, but not limited to, mammalian cancer cells such as melanoma, breast, pancreas, glioma, astrocytoma or lung cancer cells as compared to non-cancer cells. Inhibition of cancer cell growth or angiogenesis is to reduce or randomly reduce the rate of statistically significant increase in activity as compared to control treatment. Invasion into cells refers to any invasion rate into cells that is statistically significant as compared to the invasion rate into equivalent normal cells.

In some embodiments, the cupredoxin peptide is derived from, but is not limited to, azurin, pseudoazurine, plastocyanin, rustiyanin, auracyanin, stelacyanin, cucumber based protein or Laz. In certain embodiments, the azurin is Pseudomonas aeruginosa, Alkagen's Pecalis, Acromobacter silosooxydans. Dennitripacans, Bordetella bronchiceptica, Mectillomonas spp., Meningococcus, Gonococcus, Pseudomonas fluescins, Pseudomonas chlorolapis, Silera pastiosa, Ulva pertussis pertussis ), derived from enteritis vibrio. In a very specific embodiment, azurin is derived from Pseudomonas aeruginosa. In another specific embodiment, the cupredoxin peptide comprises an amino acid sequence which is the sequence of SEQ ID NOs: 1-25.

In some embodiments, the cupredoxin peptide has some of the pharmacological activity of Pseudomonas aeruginosa azurin, in particular p28. In certain embodiments, the cupredoxin peptide inhibits tumor development, reduces tumor growth or kills tumor cells in mammalian cells, tissues or animals. In some embodiments, the mammalian tumor consists of but is not limited to melanoma, breast, pancreas, glioma, astrocytoma, lung, crystal, neck, head, bladder, prostate, skin and neck cancer cells. Tumor development inhibition is the reduction of any decrease, alleviation, or increase rate of statistically significant tumor development compared to control treatment.

Cells of the invention

Provided are cells that express the selected protein upon contact with cancer cells of the invention. This feature of the invention utilizes the azurin gene of Pseudomonas aeruginosa described herein, which can be induced by contact with cancer cells. In one embodiment, the cells of the invention are derived from Pseudomonas aeruginosa and have a copy of the Pseudomonas aerugin gene in the genome, wherein the coding sequence of the azurin is replaced by the coding sequence of the target protein, upon contact with the cancer cell. The target protein is expressed. In another embodiment, the cell may be a species other than Pseudomonas aeruginosa and retains a copy of the Pseudomonas aerugin gene in its genome to express Pseudomonas aeruginosa upon contact with cancer cells. An example of this embodiment is provided in Example 3 wherein the cell retains a copy of the Pseudomonas aeruginary azurin gene in the genome, wherein the coding sequence of the azurin is replaced by the coding sequence of the target protein, such that upon contact with the cancer cell the target protein. Is expressed.

Target proteins of interest include therapeutic proteins, cytotoxic proteins and diagnostic proteins, and any protein that may be beneficial when produced in the vicinity of cancer cells. Examples of therapeutic proteins of interest include the cupredoxin proteins described herein. Methods of making such cells are known in the art and manufacturing manuals are found in those including Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Mass. (2001)). Can be. Nevertheless, the following non-limiting cell preparation and detoxification treatment methods are provided.

Exemplary Host Cell Preparation Methods

Transformed host cells can be grown in any growth inducible medium. Examples of such media include Luria Broth, 20 nM MgCl. sub.2, Luria Broth supplemented with 0.001% thiamine, 0.2% glucose; SOB medium containing 0.001% PPG (recipe provided below); 15/10 medium (recipe provided below), including but not limited to. Any other medium that one of ordinary skill in the art will recognize can be used.

Incubation temperature for growing cells varies from about 10 ° C to about 42 ° C. In one embodiment, the temperature is from about 12 ° C to about 37 ° C. In yet another embodiment, the temperature is from about 15 ° C. to about 32 ° C., and in yet another embodiment, the temperature is from about 20 ° C. to about 25 ° C. In one specific embodiment, the cells are grown at about 23 ° C.

Those skilled in the art will appreciate that growth conditions, timing of incubation may affect cell viability and transformation effects after cryopreservation.

Cells grown in shake flask culture are generally more resistant to lyophilized stress than static broth cultures. In addition, the culture age also affects the ability of the culture to survive freeze drying. Considering such lyophilization, cells obtained at late log or early stagnation stages generally exhibit maximum resistance to freeze precursors.

Therefore, shaking culture of the cells is appropriate when considering freeze drying, but other means of growth including fermentation may be used. The shake flask used can be of any type and size. In one embodiment, a baffled 2.8 liter shake flask may be used for this procedure. The timing of incubation will vary depending on the conditions used (temperature, medium, air supply) and the cell type. The air supply to the flask can vary depending on the revolutions per minute (rpm) used, the higher the rpm, the higher the air supply. In certain embodiments, the flask is generally stirred at 100-500 rpms, 200-400 rpms, 200-300 rpms but one skilled in the art can determine any other suitable range. Cells are generally grown under time and conditions at 550 nm at which optical density (OD) sufficiently reaches the range of about 0.1 to about 2.0. In one embodiment, the OD range is from about 0.1 to about 1.0. In other embodiments, the OD range is from about 0.3 to about 0.8. In yet another embodiment, the OD range is about 0.5 to about 0.8. In yet another embodiment, the OD range is about 0.5 to about 0.7. I In another embodiment, the OD ranges from about 0.6 to about 0.8. In yet another embodiment, the OD range is about 0.66 to about 0.75.

After the cells have reached the desired OD, the cells may be harvested for further processing. When the desired OD was not reached or exceeded, cells were inoculated again and the growth process was repeated until sufficient OD culture was obtained. After the cells are harvested (eg, but not limited to, centrifugation, filtration, etc.), they are optionally frozen (eg, 0 ° C.-4 ° C.-5 minutes to 2 hours). Cell collection can be carried out by centrifuging the cells to obtain cell pellets. Methods for concentrating cells include, but are not limited to, removing water from the culture, filtration or culturing the culture using size extrusion chromatography, eg, Centrtcon ™ columns (Amicon Corp .. Lexington, Mass.). This includes.

After harvesting the cells, the cells may be resuspended in competence buffer. Competence buffer refers to any solution in which cells can take exogenous DNA and establish it. Non-limiting examples of competence buffers include 50 mM CaCl ₂ ; 10 mM Tris / HCl (Maniatis. T. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY (1989)); 0.1 M MOPS (pH 6,5), 50 mM CaCl ₂ , 10 mM RbCl ₂ , 23% V / V DMSO; CCMB80 buffer (10mM potassium acetate _{pH 7.0 80mM CaCl 2 -H 2 O} , 20mM MnCl 2 4H 2 O, 10mM MgCl 2 OH 2 O, 10% glycerol - pH adjusted to 6.4 using 0.1N HCl.); TFB buffer (Liu et al., Biotechniques 8 (l): 23-25 (1990)); Ω 1776 buffer (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor, NY) 1989) Other suitable buffers include Tang et al., Nucl.Acids Res. 22 (14): 2857-2858 (1994); Chung et al., Proc. Natl. Acad Sci. USA 86: 2172- 2175 (1989); M. Dagert and SD Ehrlich, Gene 6: 23- 28 (1974); Kushner, SR, In: Genetic Engineering (HW Boyer and S. Nicosia, eds.) Pp. 17-23, Elsevier / North Holland, Amsterdam (1978); Mandel and Higa, J. MoI. Biol. 53: 159-162 (1970); US Pat. No. 4,981,797 by lessee; Hanahan, D., J. MoI. Biol. 166: 557- 580 (1983), which is hereby incorporated by reference.

Cells suspended in competence buffer are incubated at a time and temperature sufficient for the cells to take DNA. In one embodiment, the cells were incubated at low temperature (0 ° C.-4 ° C.) for about 0-3 hours. In another embodiment, the cells were incubated at low temperature (0 ° C.-4 ° C.) for about 5 minutes to 1 hour. In another embodiment, the cells were incubated at low temperature (0 ° C.-4 ° C.) for about 5-30 minutes.

After making the cells competent, cryopreservatives can be added directly to the cell suspension. In one embodiment, cells can be harvested and suspended in cryopreservatives. The concentration of cryopreservative may vary depending on the cell type, buffer used, the type of cryopreservative and other factors, and the like. Appropriate conditions may be determined by one skilled in the art without undue experimentation. When cryopreservatives are used, cryopreservatives are lowered to freezing, minimizing the effect of exogenous solution changes on the cells, infiltrating cells that are protected against solute concentration effects, or lowering the optimal cooling rate. To protect the cells during the freezing process. (F. P. Simione, Journal of Parenteral Science & Technology, 46 (6): 226-232 (1992). Of course, cryopreservatives should be non-toxic to cells.

It may be any cryopreservative and combinations thereof, but it will be apparent that the type and amount used may vary depending on the cell type and the conditions used. Cryopreservatives which may be used according to the invention include polymers such as carbohydrate and carbohydrate derivatives such as trehalozo, sucrose, maltose, glucose, sorbitol, polyethyleneamine, polyvinylpyrrolidone (PVP), picol, and the like. do. Other cryopreservatives that may be used in accordance with the present invention include acacia gum, albumin, gelatin and sugar alcohols and the like and will be readily appreciated by those skilled in the art.

After mixing the cells with the cryopreservative, the cell suspension is used for lyophilization and storage, for example, in a chilled freezing container such as a NUNC tube (Gibco BRL, Gaithersburg, Md., Cat. No. 366656), or Droplets can be added dropwise into glass vials (Wheaton, Millville, NJ.). After lyophilization, in one embodiment, the cells can be frozen from about-20 ° C to about -180 ° C. In one embodiment, the cells can be frozen at about -80 ° C to about -180 ° C. In certain embodiments, the cells can be frozen at about -80 ° C.

Methods for freezing samples from -80 ° C to about -180 ° C are known in the art. This includes storing the vial containing the cells in the -80 ° C freezer overnight (approximately 16 hours), soaking the vials containing the cells in dry ice, or soaking the vials containing the cells in a low temperature bath such as dry ice ethanol. Alternatively, a vial containing cells may be immersed in a tank containing liquefied nitrogen. Such a system is described in The Chemist's Companion: A Handbook of Practical Data, Techniques, and References, Gordon, A. J., et al, eds., John Wiley and Sons, NY (1972), which is hereby incorporated by reference.

If desired, the cells may be lyophilized using techniques known in the art. Lyophilization is the process of removing ice and / or water from frozen cells by sublimation heat at sub-zero (eg, -40 ° C to -50 ° C). Any residual moisture associated with “dried” preparations vaporizes when the temperature is raised gradually. Thus, lyophilization consists of applying the vacuum to frozen cells under conditions sufficient to substantially remove moisture and / or ice from the cells (substantially dried cells). Substantially dried cells may be stored at various temperatures (from room temperature to about −180 ° C.), and in certain embodiments, is about 4 ° C. to about −80 ° C., about −20 ° C. to about −80 ° C., and one embodiment In the example, it is stored at about -20 ° C.

One non-limiting embodiment of the lyophilization process of cells includes (a) placing a container containing lyophilized cells into a lyophilizer, wherein the lyophilizer is at a temperature of about −40 ° C. to about-50 ° C .; (b) applying a vacuum to the cells; And (c) substantially drying the cells. In one embodiment, the vacuum is less than about 100 μm, and the cells maintain (i) the temperature of the chamber at about −45 ° C. for about 2 hours, and (ii) the temperature of the chamber is about 0.1 ° C. to about 1.0 ° C. per hour. Dry at a rate from about −45 ° C. to about 100 ° C. at a rate (in one embodiment, at a rate of about 0.5 ° C. to about 0.8 ° C. per hour, and in certain embodiments, at a rate of about 0.6 ° C. to about 0.8 ° C. per hour). The cell container is sealed and stored for extended periods of time at various temperatures.

The viable host cell number of the competent cells produced by the method should be maintained at about 1 × ^{10 7} to about 1 × ^{10 9} cells / ml or more when stored at −20 ° C. for any time in the range from about 0 days to about 450 days. The cells have a transfection efficiency of at least about 1 × 10 ⁵ and preferably have at least about 1 × ^{10 9} transformants per μg (T / μg) of DNA. Suitable storage temperatures vary from room temperature to about -180 ° C. In one embodiment, the storage temperature varies from 4 ° C. to about −80 ° C. In yet another embodiment, the optimal storage temperature varies from -20 ° C to about 80 ° C. The storage period or time can vary from about 0 to about 45 days, about 0 to about 90 days, about 0 to about 150 days, about 240 to about 365 days, about 365 to about 450, and -20 ° C. Longer storage periods may be used below. Competence host cells produced by this method can be stored for at least one year while maintaining their transformation efficiency.

Exemplary Host Cell Transformation Methods.

As will be appreciated by those skilled in the art to transform host cells, the cells are mixed with the DNA molecules of interest and incubated under sufficient conditions that the cells are transformed by these DNA molecules. Any DNA molecule (eg, plasmid, phagemid, expression vector, etc.) can be used. Suitably the cells are mixed with the DNA molecules in the presence of competence buffer. Competence buffer may be added to the competence host cell prior to the addition of the DNA molecule, and the DNA molecule and the competence buffer may be added to the competence host cell simultaneously. Competence host cells are mixed with DNA molecules, although competence buffers are appropriate but any solution may be used to rehydrate the cells and mix the cells with the DNA molecules of interest. Such solutions include water, salts or any suitable buffer.

The following provides a detailed non-limiting example. Thaw about 25-200 μl cells and keep on ice. Cells should not be placed in glass tubes because the glass can absorb the DNA that is required. Carefully pipet the DNA into the cell mixture and keep the DNA volume below about 5% of the cell volume. The cells can be incubated with the DNA for about 5 to about 30 minutes. The cells are then incubated at a temperature of about 42 ° C. for about 30 seconds, then incubated on ice for about 2 minutes and then about 4 volumes of SOC medium is added, which is crucial for the success of the transformation. Can not be done. The cells are then incubated in shake at 37 ° C. for about 30 minutes to about 1 hour. After incubation, about 100-300 μl cell / DNA mixture is plated on plates with appropriate antibiotics (if necessary).

Once the cells are transformed with the desired DNA molecule, the transformed cells can be grown in growth induction medium. In general, such growth-inducing medium may include antibiotics to help the selection of transformed cells. That is, a selective marker (eg, antibiotic resistance gene) is included in the DNA to be transformed, so that the transformed cells can be selected when an antibiotic corresponding to the medium is used. Such a process is not necessary. Particularly useful methods of administration, when transformed cells (and condensed DNA) are used as treatments according to the invention, include local delivery via catheter and drug pump systems, direct delivery via topical injection or delivery via polymer Etc., but is not limited to such. In one embodiment, a “controlled dosing system” may be used, including direct and topical dosing systems. Controlled dosing systems include but are not limited to depots or pump systems such as osmotic pumps or infusion pumps, fixed speed pumps and the like. The catheter is shaped to be operably linked to a pump to deliver the cells of the invention to the target tissue site of the individual. Other delivery methods described throughout the remainder of the specification may be appropriate.

How to use

The present invention relates to the treatment of patients, in particular the treatment of patients suffering from disorders, in particular of cancer, and to methods of preventing cancer in these patients, the method comprising: CpG rich DNA and at least one cupredoxin peptide harmful to Pseudomonas aeruginosa in patients. Consisting of simultaneous administration. Such treatment may be carried out by administering one of the isolated CpG rich polynucleotides of the invention. Cancers that can be prevented or treated using the compositions of the present invention include, but are not limited to, melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, rectal cancer, neck, head, bladder, prostate, skin and neck cancer Does not. In other embodiments, the patient suffers from AIDS, malaria, inadequate vasculature, or is a higher risk group for cancer than the general population. In some embodiments the patient can be a human. In other embodiments, the patient is not a human.

Compositions comprising at least one CpG rich DNA of Pseudomonas aeruginosa and at least one cupredoxin peptide may be administered to a patient through a variety of routes and various regimes that may be known in the art. In certain embodiments, CpG rich DNA and cupredoxin peptide derived from Pseudomonas aeruginosa are administered intravenously, intramuscularly, subcutaneously, topically, orally or by inhalation. The composition can be administered to the patient using any means capable of delivering the polynucleotide and polypeptide to the patient. In some embodiments, the patient is a cancer patient, and the composition can be administered to the patient using a means capable of delivering polynucleotides and polypeptides to the tumor site. In certain embodiments, CpG rich DNA and cupredoxin peptide derived from Pseudomonas aeruginosa are administered intravenously.

In some embodiments, the methods of the present invention comprise administering to a patient at least one composition consisting of a unit dose of CpG rich DNA derived from Pseudomonas aeruginosa and a unit dose of cupredoxin peptide.

In another embodiment, the method comprises administering a unit dose of at least one composition comprising CpG rich DNA derived from Pseudomonas aeruginosa and a unit dose of at least one composition comprising a cupredoxin peptide simultaneously or in a given time range Methods to be administered within, for example, co-administration may be by administering from about 1 minute to about 60 minutes after administration of a drug or from about 1 hour to about 12 hours after drug administration.

In another embodiment, the method comprises a unit dose of at least one composition comprising CpG rich DNA derived from Pseudomonas aeruginosa and a unit dose of at least one composition comprising a cupredoxin peptide and another prophylactic or therapeutic drug. To administer one unit dose of at the same time or within a given time range, for example, from about 1 minute to about 60 minutes after administration of a drug or from about 1 hour to about 12 hours after drug administration. May be co-administered. The additional therapeutic drug may be one or more of many of those commonly used to treat the disease or side effects of the treatment of the patient. These medications include those used to treat or prevent cancer, AIDS, malaria, inadequate angiogenesis, inflammatory bowel disease, viral diseases, cardiovascular disease, peripheral vascular disease, central nervous system disease, central nervous system degeneration and Alzheimer's disease. But is not limited to this. Examples of such therapeutic agents are described in U.S. Pat. Patent Application Serial No. 10 / 047,710, Jan. 15, 2002 (U.S. Patent Serial No. 7,084,105); U.S. Patent Application Serial No. 10 / 720.603, Nov. 11, 2003 application; U.S. Patent Application Serial No. 11 / 244,105, October 6. 2005 application, U.S. Patent Application Serial No. 11 / 488,695, July 19, 2006 application; U.S. Patent Application Serial No. 1 1 / 436,592, May 19, 2006 application; U. S. Patent Application Serial No. 11 / 488,693, July 19, 2006 application; U.S. Patent Application Serial No. 11 / 436,590 May 19, 2006 application; U.S. Patent Application Serial No. 11 / 436,591, May 19, 2006 application; U.S. Provisional Patent Application Serial No. 60 / 843,388, September 11, 2006 application; U.S. Provisional Patent Application Serial No. 60.844,358, September 14. 2006 Applications, each of which is hereby incorporated by reference. Further therapeutic agents of interest can be found in the Thomson Physician's Desk Reference (Thomson Healthcare, Stamford CT. 2006) and in other references known in the art.

Drugs used to treat cancer include mechlorethamine hydrochloride, cyclophosphamide, chlorambutyl, melfaran, ifosfide, busulfan, carmustine, lomustine, semustine, streptozosin, thiotepa, dakar Vazine, methotrexate, thiogamine, mercaptopurine, fludarabine, pentastatin, cladribine, cytarabine, prourouracil, doxorubicin hydrochloride, daunorubicin, idarubicin, bleomycin sulfate, mitomycin C, actinomycin D, sapracin, sapramycin, quinocarcin, discodermoride, vincristine, vinblastine, vinorelbine tartrate, etoposide, etoposide phosphate, teniposide, paclitaxel Tamoxifen, Estramustine, Estramustine Phosphate Sodium, Flutamide, Buserelin, Lutropide, Pteridine, Levamizol, Aflacon, Interfere , Enterokin, aldes-rukin, filgrastim, sargramostin, rutushimab, BCG, tretinoin, irinotecan hydrochloride, betamethosone, gemcitabine hydrochloride, altamine, topoteca and analogs or thereof Derivatives and the like, including but not limited to.

Preferred among these types are paclitaxel, cisplatin, carboplatin, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, metopethene C, exedenasidine 743, or porphyromycin, 5-fluoro Uracil, 6-mercaptopurine, gemcitabulin, cytosine arabinoside, grapephytotoxin or grapephytotoxin derivatives such as etoposide, etoposide phosphate or teniforce, melphalan, vinblastine, Vincristine, luroshidine, vindesine and lurosine, and the like.

Examples of drugs for the treatment of cancer include the following; Epotyrone derivatives, German Patent No. 4138042.8; WO 97/19086, WO 98/22461. WO 98/25929, WO 9838192, WO 99/01124. WO 99/02224, WO 99O2514. WO 99/03848, WO 99/07692, WO 99 27890, WO 99 28324, WO 99/43653, WO 99/54330, WO 99/54318, WO 99/54319, WO 99/65913, W7O 99/67252, WO 99 / 67253, WO 00 / O0485; Cyclin dependent kinase inhibitors WO 99/24416 (U.S. Pat. No. 6.040.321): prenyl-protein transferase inhibitors WO 97/30992 and WO 98/54906; U.S. Pat, No. Substances described in 6,011,029 (compounds in the US patent can be used with other NHR modulators, which include AR modulators, ER modulators, LHRH modulators or surgical castrations, especially modulators used in cancer treatment). Including but not limited to).

Drugs for the treatment of HIV infection include reverse transcriptase inhibitors: AZT (Redovir), ddC (Zalcitabine [Hivid], Dideoxyinosine), d4T (Stavudine [Zerit]), 3TC (Ramivudine [Epivir]), non-new Cleoside Reverse Transcriptase Inhibitor (NNRTIS): Deravirdin and Nevirapine (Viramune), Protease Inhibitors: Ritonavir (Lorvir), Lopinavir and Ritonavir Complex (Kaietra), Saquinavir (Invirase), Indinavir sulfate (Crixivan), Amprenavir (Agenerase), and Nelfinavir (Viracept). Currently, several drug complexes, called highly active anti-retviral therapy (HAART), are used to treat HIV patients.

Drugs to treat malaria include progunanil, chloroproguanyl, trimethoprim, chlorovinin, mefloquinine, lumepantrin, atobaquinone, pyrimethamine-sulfadoxin-dopason, halophantrin, quinine, Quinidine, Amodiaquinine, Amopyroquinine, Sulfonamide, Artemisinin, Artemeter, Artesonate, Primaquine, Pironaridine, Proguanyl, Chloroquine, Mefloquinine, Pyrimethamine-Sulfadoxin , Pyrimethamine-dopason, halophantrin, quinine, proguanil, chloroquine, mefloquinine, 1,16-hexadecamethylenebis (N-methylpyrrolidinium) diblomide, and complexes thereof But is not limited to this.

Drugs for treating inflammatory bowel disease include aminosalicylates such as sulfazaldine, olsarazine, mesalamine, Pentasa) and Valsalazide (Colazal); Corticosteroids such as predinison, Medrol, methylpredinizolone, hydrocortisone, Budesonide (Entocort EC); Immunomodulators such as azathioprine (Imuran), 6-mercaptopurine (6-MP. Purinethol) and cyclosporin A (Sandimmune, Neoral); Antibiotics such as metronidazole and ciprofloxacin (Cipro); Biological therapeutic agents such as Remicade; And other therapeutic agents such as but not limited to taclolimus (FK506) and mycophenolate mofetil and the like.

Drugs for the treatment of HIV infection include reverse transcriptase inhibitors: AZT (Redovir), ddC (Zalcitabine [Hivid], Dideoxyinosine), d4T (Stavudine [Zerit]), 3TC (Ramivudine [Epivir]), non-new Cleoside Reverse Transcriptase Inhibitor (NNRTIS): Deravirdin and Nevirapine (Viramune), Protease Inhibitors: Ritonavir (Lorvir), Lopinavir and Ritonavir Complex (Kaietra), Saquinavir (Invirase), Indinavir sulfate (Crixivan), Amprenavir (Agenerase), and Nelfinavir (Viracept).

Drugs for the treatment of viral diseases include acyclovir, varicella zoster immunoglobin (VZIG), pejiinterferon, ribavirin, azovirx, Valtrex, famvirvir, amantadine, rimantadine , But not limited to zanamivir, oseltamivir and alpha interferon.

Drugs for the treatment of cardiovascular diseases include anticoagulants, antiplatelet substances, thrombotic substances, adrenergic blockers, adrenergic stimulants, alpha / beta adrenergic blockers, angiotensin converting enzymes (ACE) inhibitors, and angiotensin converting enzymes (ACE) inhibitors. Substances and calcium channel blockers, angiotensin converting enzyme (ACE) inhibitors and diuretics, angiotensin II receptor antagonists, calcium channel blockers, diuretics (carbonic anhydrase inhibitors, loop diuretics, potassium-preservatives) Diuretics, thiazides and related diuretics), vasodilators, vasodilators, and the like.

Drugs for treating peripheral vascular diseases include pentociphylline (Trental), oral methylxanthine derivatives and cyrostazol (PIetal), phosphodiesterase III inhibitors; Antiplatelet agents / antithrombotic agents such as aspirin; Anticoagulants such as heparin and warfarin (Coumadin); Cholesterol lowering drugs such as niacin, statins, fibrates, lopid tablets (gemfibrozil; Parke-Davis); Fenofibrate (Abbott), bile acid sequestrants, colestid tablets (mieronized colestipol hydrochloride; Pharmacia and Upjohn); welchol tablets (colesevelam hydrochloride; Sankyo); Calcium channel blockers; Vitamins and dietary supplements such as folic acid, B-6, B-12, L-arginine and omega-3 fatty acids; HMG-COA Reductase Inhibitors such as advicor tablets (Niacin / Lovastatin; Kos); altocor extended-release tablets (lovastatin; Andryx labs); lescol capsule (fluvastatin sodium; Novartis &Reliant); lipitor tablets (atorvastatin; Parke-Davis and Pfizer); mevacor tablets (lovastatin; Merck); pravachol tablets (Pravastatin sodium; Bristol-Myers Squibb); pravigard PAC tablets (buffered Aspirin and Pravastatin Sodium; Bristol-Myers Squibb); zocor tablets (Simvastatin; Merck); Nicotinic acid substances such as advicor tablets (Niacin / Lovastatin; Kos (HMG-COA Reductase inhibitor)); niaspan (niacin; Kos); Other substances include, but are not limited to, zetia tablets (Emertis / Schering Plough).

Drugs for treating central nervous system diseases include psychotherapeutics such as various benzodiazepine preparations and complexes, anti-anxiety agents, antidepressants (monoamine oxidase inhibitors (MAOIs), selective serotonin reuptake inhibitors (SSRIs, tricyclic antidepressants), antipharmaceuticals (antimanic), anti-aircraft drugs, antipsychotics, psychostimulants, and obsessive-responsive disorder modulators, migraine preparations such as beta adrenergic blockers, isometheptene and serotonin receptor agonists and depakote tablets (Divalproex sodium; Abbott) and excedrin Other migraines, including the active ingredient in acetaminophen (BMS Products); Sedatives and sleeping pills; Anticonvulsants; And includes but is not limited to pimozide. Drugs for treating Parkinson's disease include, but are not limited to, anticholinergic agents, catechol-o-methyltransferase inhibitors, dopamine substances, and monoamine oxidase (MAO) inhibitors.

Drugs for the treatment of central nervous system (CNS) degenerative diseases include: Drugs for treating multiple infarctions include iavonex (interferon beta-1b; Biogen Neurology); Betaseron for SC injection (modified form of Interferon beta-lb; Beriex); Injectable copaxone (Giatiramer Acetate: Teva Neuroscience); depo-raedror injectable suspensions (Methylprcdnisolone acetate; Pharmacia &Upjohn); Novantrone for injection concentration (Mitoxantrone supplied as mitoxantrone hydrochloride; Serono); Orapred Oral Solution (Predinizolone Sodium Phosphate Oral Solution: Ascent): Includes, but is not limited to, the active ingredient of Rebif injection (interferon beta-1a; Pfizer & Serono). Drugs for treating Huntington's disease include sedatives such as Klonopin; Antipsychotic drugs such as haloperidol (Haldol) and clozapine (Clozaril); Fluoxetine (Prozac, Sarafem), sertraline (Zoloft), nortriphytiline (Aventyl, Pamelor). And lithium (Eskalith, Lithobid).

Drugs for the treatment of Alzheimer's disease include aricept tablets (Donepezil Hydrochloride; Eisai or Pfizer); exelon capsules (rivastigmine (hydrogen tartrate salt); Novartis); exelon oral solution (rivastigmine tartrate; Novartis); reminyl oral solutions (galantamine hydrobromide; Janssen) or reminyl tablets (galantamine hydrobromide; Janssen) and the like.

Chemopreventive drugs of interest include tamoxifen, aromatase inhibitors such as letrozole and anastrozole, retinoids such as N- [4-hydroxyphenyl] retinamide (4- HPR, fenretinide), nonsteroidal anti-inflammatory agents (NSAIDs) such as aspirin and sulindac, celecoxib (COX-2 inhibitors), defluoromethylornithine (DFMO), ursodeoxycholic acid, 3-hydroxy -3-methylglutaryl coenzyme A reductase inhibitor, EKI-785 (EGFR inhibitor), bevacizumab (antibody to VEGF-receptor), cetushimab (antibody to EGFR), retinol such as vitamin A , beta-carotene, 13-cis retinoic acid, isotretinoin and retinyl palmitate, α-tocopherol, interferon, oncolytic adenovirus dll520 (ONYX-015), zephytinib, etretinate, finasteride, indole-3- Carbinol, resveratrol, cologenenoic acid, raloxyphene and And the like PIFF Raj, but not limited to, sikijineun.

The method of the invention further comprises a method of administering a cell of the invention. Such methods include methods of treating patients, especially those with disabilities, particularly cancer patients, and diagnosing cancer in the patients. In some embodiments, a cancer patient can be treated by administering a cell that expresses azurin upon contact with the cancer cell or that expresses a therapeutic or cytotoxic target protein. In other embodiments, the patient is a patient, including but not limited to cancer, such as melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, rectal cancer, neck, head, bladder, prostate, skin and neck cancer . In another embodiment, the method comprises a method of diagnosing cancer in a patient consisting of administering a cell expressing the diagnostic protein upon contact with the cancer cell and sensing the diagnostic protein at the bloodstream or site of expression.

The methods of the present invention can be administered to a patient using any suitable means including, but not limited to, intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, suppositories, vitreous injection, and oral. In certain embodiments, the cells are injected intravenously.

CpG  rich DNA  Or a pharmaceutical composition comprising a cell

Pharmaceutical compositions comprising CpG rich DNA derived from cells or Pseudomonas aeruginosa of the present invention may be prepared by any conventional method such as conventional mixing, lysis, granulation, dragee making, emulsification, capture, capture, or lyophilization. It can manufacture. Substantially pure or medicinal grade CpG rich DNA derived from the cells of the present invention or Pseudomonas aeruginosa can be directly complexed with known pharmacologically acceptable carriers. Such carriers may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like. Other excipients include flavors, colorants, detackifiers and thickeners and other acceptable additives, adjuvants or binders and the like. In some embodiments, the pharmaceutical formulation is substantially free of preservatives. In other embodiments, the pharmaceutical formulation may include at least one preservative. General methods of the dosage forms can be found in Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems (Lippencott Williams & Wilkins. Baltimore MD (1999)).

Pharmaceutical compositions comprising CpG rich DNA derived from cells or Pseudomonas aeruginosa of the invention can be administered in a variety of ways, including by injection (dermal, subcutaneous, muscle, peritoneal and similar), inhalation, topical administration, suppositories, transdermal patches or oral. May be administered. General information on drug delivery systems can be found in Ansel et al., Id. In some embodiments, pharmaceutical compositions comprising CpG rich DNA derived from cells or Pseudomonas aeruginosa of the invention can be formulated and used directly as an injectable for subcutaneous and intravenous injection. In particular, injection preparations may be advantageously used to treat patients suitable for therapy.

Pharmaceutical compositions comprising CpG rich DNA derived from cells or Pseudomonas aeruginosa of the invention may optionally be administered orally after mixing with a protective material or similar coating, such as polypropylene glycol.

When administered via injection, a pharmaceutical composition comprising CpG rich DNA derived from the cells of the present invention or Pseudomonas aeruginosa may be formulated with an aqueous solution, in particular Hanks' solution, Ringer's solution or physiological salt buffer. These solutions include formulary agents such as suspending, stabilizing or dispersing agents. Alternatively, CpG rich DNA derived from Pseudomonas aeruginosa is powdered and can be reconstituted prior to use with a suitable vehicle, eg, sterile pyrogen-free water, before use. In some embodiments, the pharmaceutical composition does not include an adjuvant or any other substance added to enhance the immune response stimulated by the polynucleotide. In some embodiments, the pharmaceutical composition includes a substance that inhibits an immune response to the polynucleotide.

If administration is by intravenous fluid, the pharmaceutical composition comprising CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the invention may be composed of crystals or colloids. Crystals used herein are aqueous mineral salt solutions or aqueous solutions of other water soluble molecules. Colloids here include larger insoluble molecules such as gelatin. Intravenous fluid may be sterile.

Examples of crystalline fluids that can be used for intravenous administration are conventional salts (0.9% sodium chloride solution), Ringer's lactate or Ringer solution or 5% dextrose solution in water called D5W, as shown in Table 1. Including but not limited to.

Conventional Crystal Solution Composition solution Another name [Na ⁺ ] [CT] [Glucose] D5W 5% dextrose 0 0 252 2/3 & 1/3 33% dextrose / 0.3% salt 51 51 168 Half normal saline 0.45% NaCl 77 77 0 Normal salt 0.9% NaCl 154 154 0 Ringer lactate * Ringer solution 130 109 0

* Ringer's lactate and includes a 28mmol / L lactate, 4mmol / LK ⁺ and 3mmol / L Ca ^{2 +.}

When administered via inhalation, the pharmaceutical composition comprising CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the invention may be in the form of an aerosol spray from a pressurized pack or in a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane. , Aerosol spray can be delivered from the nebulizer using carbon dioxide or other suitable gas. In the case of a pressurized aerosol, a unit dose may be provided by providing a valve that delivers a fixed amount. When using an inhaler or pulverulent, it can be made to contain a mixed mix of polynucleotides and suitable powders such as lactose or starch in cartridges and capsules such as gelatin.

When administered via topical administration, the pharmaceutical composition comprising CpG rich DNA derived from the cells of the present invention or Pseudomonas aeruginosa can be formulated in solutions, gels, ointments, creams, jelly, suspensions and similar forms known in the art. have. In some embodiments, the administration consists of a transdermal patch. When administration is by suppository (eg rectal or vaginal), pharmaceutical compositions comprising CpG rich DNA derived from cells or Pseudomonas aeruginosa of the present invention may be formulated into a composition comprising a conventional suppository base.

When the administration is oral, the pharmaceutical composition comprising CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the present invention may be prepared by combining the CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the present invention with a pharmacologically acceptable carrier known in the art. It can be prepared immediately. Mannitol, lactose, magnesium stearate and similar solid carriers may be used; Such carriers may be used in the form of tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like which can be taken orally by an individual to be treated with a pharmaceutical composition comprising CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the invention and It should be prepared in a similar form. In the case of oral solid preparations such as powders, capsules and tablets, suitable excipients may include fillers such as sugars, cellulose preparations, granulates, binders. Other conventional carriers as known in the art include bacterial capsular polysaccharides, dextran or genetically engineered vectors. In addition, delayed-release preparations comprising CpG rich DNA derived from Pseudomonas aeruginosa allow release of CpG rich DNA for a delayed time, and CpG rich DNA of Pseudomonas aeruginosa, for example, when the delayed release preparation is not used, has a therapeutic effect, for example. May be degraded by proteases and simple hydrolysis or removed from the individual's system before exerting or enhancing.

In various embodiments, pharmaceutical compositions include but are not limited to carriers and excipients (buffers, carbohydrates, mannitol, amino acids such as proteins, polypeptides or glycines, antioxidants, bacteriostatic agents, chelating agents, suspending agents, thickening agents and / or preservatives). Other pharmaceutically acceptable auxiliaries, wetting agents, and the like required to be close to physiological conditions such as water, oils, salt solutions, water soluble dextrose and glycerol solutions, buffers, tonic modifiers, and the like. Include. While any suitable carrier known to those skilled in the art can be used to administer the compositions of the present invention, it will be appreciated that the carrier type may vary depending upon the mode of administration. The compound may also be encapsulated in liposomes using techniques known in the art. Biodegradable microspheres can also be used as carriers for the pharmaceutical compositions of the present invention. Suitable biodegradable microspheres are described in U.S. Patent Nos. 4,897,268; 5,075,109; 5,928.647: 5,811,128; 5.820.883; 5.853,763: 5,814.344; 5,942.252 is known.

Pharmaceutical compositions comprising CpG rich DNA may be stabilized or sterile filtered by conventional known stabilization techniques. The resulting aqueous solution is packaged or lyophilized for use where the lyophilized formulation is combined with a sterile solution prior to administration.

CpG  rich DNA  Or administration of cells

CpG rich DNA derived from a cell or Pseudomonas aeruginosa of the invention may be administered in the form of a pharmaceutical composition or by any suitable route, such as oral, buccal, inhaled, sublingual, rectal, vaginal, urethral, nasal, topical, transdermal (eg , Via the skin) or extra-intestinal (including vein, muscle, subcutaneous, coronary) or via vitreous administration. Pharmaceutical preparations thereof may be administered in any effective amount to achieve the desired purpose. More specifically, the present invention is administered in a prophylactic or therapeutically effective amount. In certain embodiments, the prophylactic or therapeutically effective amount is generally about 0.01-20 mg / day / kg of body weight. Compounds comprising CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the present invention may be used alone or in combination with other active ingredients to be useful in treating abnormalities, in particular in treating or preventing cancer. Appropriate doses may vary depending on such compounds as used, including the CpG rich DNA derived from Pseudomonas aeruginosa cells, the host, route of administration, disease characteristics and severity. However, a daily dosage of 0.01-20 mg / kg has generally been shown to yield satisfactory results in humans. For example, a daily, weekly, monthly or continuous dose specified in humans is from about 0.7 mg to about 1400 mg of a compound comprising CpG rich DNA derived from the cells or Pseudomonas aeruginosa of the present invention. The daily dose is 1 to 12 separate unit doses per day. Alternatively, the dose may be administered once every two days, once every three days, once every four days, once every five days, once every six days, from weekly and daily to up to 31 days or more. Or the dose may be administered continuously using the patch in iv administration and in a similar manner.

Depending on the patient's condition, the attending physician determines the correct preparation, route of administration, and dosage. Dosage and dosing intervals are adjusted individually to provide plasma levels of CpG rich DNA and cupredoxin peptides derived from Pseudomonas aeruginosa of the present invention to maintain a prophylactic or therapeutic effect. Generally, the desired CpG rich DNA from Pseudomonas aeruginosa is administered in admixture with pharmaceutical carriers selected according to the desired route of administration and standard pharmacopeias. In the case of cells, the dosage and interval of administration are adjusted individually to provide a target protein or azurin level at the cancer cell site to maintain a prophylactic or therapeutic effect or sufficient for diagnostic presence.

CpG  rich DNA  Or containing cells Kit

In one aspect, the invention provides a kit or regimen comprising one or more of the following components in a package or container: (1) a pharmaceutical composition comprising CpG rich DNA harmful from Pseudomonas aeruginosa; (2) a pharmaceutical composition comprising at least one cupredoxin peptide; (3) additional prophylactic or therapeutic drugs; And (4) devices, such as syringes, nebulizers, for administering biologically active substances to a patient. In another aspect, the invention provides a kit or regimen comprising one or more of the following components in a package or container: (1) a cell of the invention; (2) A device for administering cells to a patient.

When the kit is supplied, different components of the composition may be packaged in separate containers and mixed just prior to use. The separate packaging of these ingredients allows for long term storage without losing the function of the active ingredient. The components included in the kit are supplied to any container that preserves the half life of the different ingredients and is not absorbed or modified by the material of the container. For example, sealed glass ampoules include lyophilized CpG rich DNA, or buffer, packaged under a neutral, non-reactive gas such as nitrogen. The ampoule may be composed of any suitable material, such as glass, organic polymers such as polycarbonate, polystyrene, etc., any other material generally used to hold ceramics, metals or similar reagents. Another example of a suitable container constitutes an ampoule, a sheath, such as a foil-lined interior, including a simple bottle made of a material such as aluminum or alloy. Other containers include test tubes, vials, flasks, bottles, syringes or the like. The container includes a bottle with a stopper that can be drilled with a sterile inlet port, for example a hypodermic needle. Other vessels may have two compartments separated by membranes that can be removed immediately, and the membranes are removed to mix the components. The removable film can be glass, plastic, rubber, and the like.

Kits may include instructional materials. Instructions may be printed on paper or other substrate or supplied on electronic-readable media, such as floppy disks, CD-ROMs, DVD-ROM-zip discs, video data, audio data, flash memory devices, and the like. Detailed instructions are not physically included in the kit and the user can receive content from an Internet web page or e-mail or the like specified by the manufacturer or distributor.

With reference to the following specific embodiments, the present invention will be more clearly understood. The examples are for illustrative purposes and are not intended to limit the scope of the invention thereto. Modifications and substitutions of equivalents may also be considered. Although specific terms have been used, such terms are for illustrative purposes and are not intended to be limiting. Modifications and various other embodiments of the present invention do not depart from the scope of the present invention.

Example  One. MCF Pseudomonas aeruginosa was cultured in the medium in the presence of -7 cells. Azurin  Release

Azurin of Pseudomonas aeruginosa is known to be secreted into the growth medium at the periplasmic protein or at the late stage of growth. Zaborina et ai, Microbiology 146: 2521-2530 (2000). Its extracellular release relies on quorum sensing at high cell densities and is regulated by GacA or two small RNA products. RsmY and RsmZ, Kay et al. J, Bacteriol. 188: 6026-6033 (2006). Experiments were conducted to determine whether Pseudomonas aeruginosa releases azurin into extracellular medium when contacted with cancer cells. A mucoid isolate strain 8821 of Pseudomonas aeruginosa and its spontaneous non-mucoid (non-alginate secretion) derivative 8822 were used from cystic fibrosis patients. Darzins et at. (J. Bacteriol, 161: 249-257 (1985)). When grown on a minimal medium supplemented with histidine, azurin is released into the growth medium at about mid-exponential growth time after about 4 hours of strain 8822 sms growth. Azurin can not be seen in the growth medium for very early on a rad or exponential of about 2 hours of growth. Mucoid strain 8821 lacks azurin secretion even in late exponential or early stagnant phases. Mucoid strains are known to lack extracellular protein secretion due to the massive release of exopolysaccharide alginates. Ohman and Chakrabarty. Infect. Immun. 37: 662-669 (1982); Woods et al. J. Infect. Dis. 163: 143-149 (1991).

1.6X10 ⁴ of human breast cancer cell line MCF-7 To ⁴ × 10 ⁵ cells were added to 8821 and 8822 cells (OD of about 660 nm at about 0.3), grown for several hours, then the cells were centrifuged and the growth medium filtered using a 0.22 μm filter followed by anti-adjuvant Azurin levels were measured using Western blotting with antibodies. Punj et al, Oncogene 23: 2367-2378 (2004). Cells without MCF-7 cells but with the same amount of growth medium were used as controls.

Addition of MCF-7 cells as mucoid strain 8821 or non-mucoid strain 8822 has little effect on the growth rate of Pseudomonas cells (FIG. 1A, a). Interestingly, however, MCF-7 cells induced release of significant amounts of azurin into extracellular growth medium within 20 minutes (measured by Western blotting (FIGS. 1A, c)) but only in 8822. Without MCF-7 cells There was no azurin release for an hour or hours in the mucoid strain 8821, which is known to be deficient in extracellular proteins (Figures 1A, b and c.) Azurin secretion quantization of Weston blotting using a standard curve (FIG. 1B) Similar results were obtained for another human cancer cell melanoma Mel-2 (data not shown) Azurin after 20-30 minutes exposure to cancer cells in the case of MCF-7 and Mel-2 Although release was observed, there was no release in the absence of cancer cells, thereby strongly indicating that strain 8822 is capable of detecting the presence of human cancer cells, releasing azurin in response to their presence. The presence of the MCF-10A normal breast epithelial cells reconciliation by a similar secretion was is to appear is delayed about 40 minutes very lean emission level was dependent on the concentration of the cancer cells; 1x10 ⁸ / ㎖ 8822 cells 2x10 ⁶ / ㎖ cancer When exposed to, much more azurin was released into the growth medium than when exposed to 1.6 × 10 ⁶ / ml (<0.1 nM azurin) or 8 × 10 ⁴ / ml (0.7 nM azurin), thereby exposing MCF-7 cells. Cyzarine release indicates that it is dose-dependent.

To confirm that mucoid strain 8821 did not lack azurin production, intracellular levels of azurin were prepared by making cell lysates and separating azurin using SDS-PAGE using a fixed amount of lysate protein (50 μg). Were measured in strains 8821 and 8822 (FIG. 1C). Both strains 8821 and 8822 explained the presence of intracellular azurin (periplasmic protein), secretion observed only in non-mucoid cells. About 6-8% intracellular for 60 min incubation time in quantization of azurin in intracellular and extracellular aliquots of grown strain 8822 with or without MCF-7 from the standard curve (FIG. 1B) It is explained that azurin was released to external media.

Example  2. Cell Without melting MCF - 7 is  From Pseudomonas aeruginosa Azurin  Induce release.

A broad host of lacZ genes that encode β-galactosidase that function to determine whether additional cancer cells can induce lysis from Pseudomonas aeruginosa and release azurin and other proteins into the growth site. A range of plasmid pQF47 was introduced into strain 8822. The release of azurin and β-galactosidase in the growth medium with and without MCF-7 cells was evaluated for 60 minutes.

There was no measurable release with LacZ extracellular growth medium in the presence or absence of MCF-7 (control) during the 60 minute incubation period (FIG. 2A). Examination of the cell lysate demonstrates the presence of intracellular LacZ in the presence or absence of cancer cells (FIG. 2A), indicating little cell lysis and little release of intracellular LacZ into the external growth medium. However, the test for presence of azurin in growth medium demonstrates that azurin is released and detected in the presence of MCF-7 and that in the absence of MCF-7, azurin is rarely detected (FIG. 2B), which indicates that MCF- Explicitly explain the role of 7 in inducing azurin secretion rather than LacZ. As expected, Pseudomonas aeruginosa 8822 lacks a functioning LacZ gene in the measurement of intracellular LacZ of strain 8822 alone and strain 8822 (8822; pQF47) with pQF47 plasmid, but lacks LacZ protein production. Explain that it produces (Fig. 2C). The presence of azurin in external media was confirmed not by cell lysis but by the fact that mucoid strain 8821 generally lacks protein release and also lacks azurin release (FIG. 1A), and cell lysis results in azurin as a growth medium. Make sure you release it.

Concentrated filtered growth medium of MCF-7 was used to determine whether Pseudomonas aeruginosa 8822 responded to any proliferative metabolite from cancer cells. In this situation no azurin release was observed, which explains that the azurin release is contact-dependent and energy independent of secretion. This method of secretion is distinct from the release of azurin upon exposure to cancer cells from other protein releases. Kostakioti et al., J. Bacterid. 187: 187: 4306-4314 (2005); Thanassi et al, MoI. Membr. Biol. 22: 63-72 (2005).

Example  3. From E. coli MCF -7 cells specifically Azurin  Induce release.

Pseudomonas aeruginosa is a periplasmic protein between the cell wall and the cytoplasm. When the Pseudomonas aeruginosa gene is expressed in Escherichia coli JM109, the resulting azurin protein is found in the space between the cell wall and the cytoplasm and purified from this space of E. coli. Goto et al, MoI. Microbiol. 47: 549-559 (2003). Another cell wall and periplasmic protein of Pseudomonas aeruginosa is cytochrome c ₅₅₁ , a partner of electron transfer isazinin in vitro, and the periplasmic space of E. coli JCB7120 grown in an anaerobic environment. It can be purified from. Id.

The secretion of _azurin and cytochrome c ₅₅₁ with or without MCF-7 was investigated from Pseudomonas aeruginosa and E. coli strains. In this study, exposure to MCF-7 breast cancer cells ruptured the outer membrane of bacteria, allowing the release of arbitrary cell wall and periplasmic proteins and specificity for azurin secretion. The investigation also confirmed whether the release of azurin is specific for Pseudomonas aeruginosa or occurs in other bacteria, such as E. coli. The levels of intracellular or extracellular cytochromes of Pseudomonas aeruginosa 8822 at aerobic growth conditions were too low to be detected by Western blotting. However, both azurin and cytochrome c ₅₅₁ were clearly detected in the extract of E. coli overexpressing the protein (Fig. 3A, B, time 0 *). Similar to Pseudomonas aeruginosa, E. coli cells carrying the Pseudomonas aeruginosa azu gene significantly release azurin into extracellular medium, but only in the presence of cancer cells. Interestingly, when E. coli cells with periplasmic cytochrome c ₅₅₁ were exposed to MCF-7, cytochrome c ₅₅₁ was detected very low in extracellular growth medium (FIG. 3B), which is MCF-7. Exposure to cells specifically induces _azurin , but does not induce cytochrome c ₅₅₁ from E. coli when intracellular cytochrome c ₅₅₁ is clearly detected (FIG. 3B).

Example  4. from Pseudomonas aeruginosa or E. coli Azurin  Emission is energy independent.

At two different concentrations (50 μM and 250 μM for P. aeruginosa 8822; 250 μM for E. coli JM109) before exposing the bacteria to MCF-7 cells to confirm that azurin release from Pseudomonas aeruginosa 8822 or E. coli JM109 requires energy. Incubation was carried out for 1 hour in the presence of protonopore carbonyl cyanide chlorophenylhydrazone (CCCP). CCCP is uncoupler to the oxidative phosphorylation reaction and inhibited bacterial growth at the concentration used. (Fig. 3E; left panel, P. aeruginosa 8822; right panel, E. coli JM109). Nevertheless, CCCP has little effect on azurin secreted by Pseudomonas aeruginosa 8822 (FIG. 3C) or E. coli JM109 (FIG. 3D), indicating that azurin secretion is energy independent.

Example  5. MCF -7 cells from Pseudomonas aeruginosa 15 kd DNA  Induced release.

Bacterial DNA is known to be released or metastasized during conjugation in response to cellular needs using a type IV secretion system. Cascales and Christie, Nature Rev. Microbiol. 1: 137-149 (2003). In such a system, direct delivery of DNA and proteins from the cell wall to the vagina, as well as the cytoplasm of donor bacteria, can occur to the target host cell. However, the presence of host cells or contacting host cells is not mandatory because the release of protein or chromosomal DNA into growth ratios occurs by the Type IV system without any host cells. Burns, Curr. Opin. Microbiol. 2: 25-29. 1999; Hamilton et al, MOL Microbiol. 55: 17044721 (2005). It is known that DNA released from Pseudomonas aeruginosa contributes to pre-inflammatory processes during infection or biofilm formation in the lungs of cystic fibrosis kanji. Delgado et al. Infect. Immun, 74: 2975-2984 (2006): Whitchurch et a U Science 295: 1487 (2002). No specific release of CpG-rich extrachromosomal DNA has been reported.

In an attempt to determine whether Pseudomonas aeruginosa 8822 releases not only azurin as a growth medium but also genomic DNA, the growth medium of strain 8822 was tested for 1 or 2 hours, with almost no azurin when exposed to MCF-7 cells. I couldn't see it. To detect the presence of any nucleoprotein complexes, three times the volume of normal isopropanol was added to the growth medium and incubated at −80 ° C. for 1 hour. Nucleoprotein pellets were centrifuged, resuspended in double distilled distilled water and passed through a Qiagen DNA preparation kit (Qiagen. Inc .. Valencia, Calif.) Before eluting with double distilled water. There was no DNA detection at 0 hours (start of experiment), but a specific DNA band of about 15 kb size was detected at 1 and 2 hours with and without MCF-7 (FIG. 4A).

In the presence of cancer cells, the amount of DNA was higher, which intensified the release of azurin, and in contrast to azurin, DNA was detected even in the absence of cancer cells, which was described by PicoGreen (Invitrogen, Inc., Carlsbad, CA). And PCR reactions are presumably due to the higher sensitivity to detection. Interestingly, other significant DNA bands are not seen, indicating that they are very reproducible under repeated experimental conditions, indicating that they are not random products of chromosomal DNA cleavage.

Example  6. 15 with emission profiles kb DNA  Is Extrachromosomal DNA Confirm that

Genomic islands with virulence associated genes are well known to Pseudomonas aeruginosa. Kiewitz et al. Microbiology 146: 2365-2373 (2000); He et al., Proc. Natl. Acad. Sci. 101: 2530-2535 (2004); Klockgether et al, J. Bacterid. 186: 518-534 (2004); Kulasekara ct al, J. Bacteriol. 188: 4037-4050 (2006). Biodegradable gene clusters that confer the advantage of selective growth in bioforeign-contaminated environments are known as mobile gene elements that are often associated with phage genes in Pseudomonas aeruginosa. van der Meer et al, Arch. Microbiol. 175: 79-85 (2001). The single gene island, PAGI-I, has at least two different DNA fragments, one that is responsible for the translation of reactive oxygen species, one that has a G + C content of 63.7%, and the other that is 54.9%. It was confirmed to exist in the sieve. Liang et al. J. Bacteriol. 185: 843-853 (2001).

Similar to azurin release, the release kinetics of this DNA fragment can be seen to exist extracellularly as early as within 5 minutes even in the absence of MCF-7 cells (FIG. 4B). Under these conditions no other DNA bands could be seen. The release kinetics studies of the release kinetics of azurin and 15 kb fragments in the absence of any other chromosomal DNA fragment upon exposure to MCF-7 cells illustrate a similar release time process (FIG. 4C), such as genomic islands where DNA is probably required in the horizontal direction. Extrachromosomal fragments that fall out of the chromosome loop.

Example  7. From Pseudomonas aeruginosa 15 kb  Fragment release CpG -rich DNA to be

DNA was subjected to various proposed enzyme endonuclease digestions to examine the released DNA properties. Interestingly, MSP-I and PvuI, known to cleave between G and C, cleave a lot of DNA, indicating that the DNA is high in G-C (FIG. 4D). When sequencing partially or mechanically cleaved fragments of these restriction enzymes were sequenced and compared to the homology of the sequences in the database, several DNA sequences present and absent in the Pseudomonas aeruginosa PAO genome can be seen (FIG. 7). SEQ ID NOS: 26-62, which refer to the strain 8822 from which DNA released from genomic islands was derived. There was no plasmid DNA isolated by conventional separation methods. The sequence of interest present in the CpG rich DNA is a cytosine stretch with many CpG nucleotide sequences in the sequence (FIG. 5A).

Example  8. 15 kb CpG  rich DNA  In the band Azurin and  Have a similar sequence.

There is an interesting sequence in the azurin gene, which resembles the azurin gene of gonococcus, with 95% nucleotide homology. Amino acid sequence comparison is shown in Figure 5B. The Neisserial azurin gene has an additional DNA sequence at the 5 'end, encoding the 39 amino acid H.8 epitope at the N-terminus of the azurin, and using CpG rich DNA as a template. And full length genes of gonococcal H.8 and azurin gene laz were used as well as the azurin gene sequence. Hong et al, Cell Cycle 5: 1633-1641 (2006). These three fragments are amplified using PCR to explain the presence of these components of gonococcal laz genes in CpG rich DNA.

Example  9: released CpG  rich DNA Is TLR9 Mediated NF - kB To Activate it  Has antitumor properties

CpG deoxyoligonucleotides have been reported to have antitumor properties. Krieg, Nature Med. 9: 831-835 (2003): Krieg, Curr. Oncol. Rep. 6: 88-95 (2004). To confirm that the 15 kb CpG-rich released from Pseudomonas aeruginosa 8822 had properties similar to CpG synthetic oligodeoxynucleotides (ODNs), we tested whether the DNA has the ability to activate NF-kB in a TLR9 dependent manner. HEK293 cells deficient in TLR9 were transfected in the presence of TLR9 expressing plasmids. Kandimalla et al, Proc. Natl Acad Sci USA i 02: 6925-6930 (2005). A pNIFty plasmid expressing SEAP (secreted embryo alkaline phosphatase) was used under the control of an NF-kB inducible ELAMl mixing promoter (Invitrogen. Inc., Carlsbad, Calif.). SEAP expression was measured after NF-kB activation in supernatants of transfected HEK293 cells using the SEAP Reporter Test Kit (Invitrogen. Inc., Carlsbad. CA). SEAP reporter assays were performed as described in Schindler and Baichwal (MoI. Cell Biol. 14: 5820-5831 (1994)).

There was little expression of NF-kB-promoter induced SEAP in TLR9 deficient HEK293 cells (FIG. 6A). However, SEAP activity increased with increasing amount of CpG-rich 15 kb DNA in TLR9-expressing-HEK293 cells, indicating that the DNA activates the NF-kB promoter only in TLR9 pluripotent cells (FIG. 6A).

Expression of TLR9 was measured in various cancer cells to confirm that activation of TLR9-dependent NF-kB resulted in the death of tumor cells. Basically all cancer cell lines, prostate cancer DU145, breast cancer MCF-7 and lung cancer H23 and A549 expressed TLR9 and other Toll-like receptors such as TLR4 (FIG. 6B). When CpG-rich DNA was incubated for 12 hours and 24 hours with increasing concentrations of lung cancer cell A549, cell death, although limited, was increased, indicating that CpG-rich DNA has low anticancer activity (FIG. 6C). .

Example  10. Effect study

We will run a double-blind, placebo controlled study for six months. A total of 80 cancer patients aged 45-66 years will be screened for the study. Primary recruitment will be made through a doctor's recommendation. Successfully preselected subjects will be contacted by phone and invited to the selection orientation. Prospective subjects are orally and writtenly informed of the study process and regulations for participation in the study. Written consent is obtained from each participant during the screening / orientation process, and medical records are obtained and forwarded to the Project Cancer Specialist. Participants will be assigned a study ID number. The following information will be collected during the screening process:

Details (Demographics): name, address, contact, primary charge, finishing the year cancer specialist, date of birth, race, profession, education end.

Medical history : Current and past medical records (cancer status, drugs, medications, hospitalization, surgery, allerty, smoking, drinking, contraindications)

Basic physical examination ; Check your height, weight, blood pressure, pulse, chest, lungs, heart abdomen.

Blood sample ; Pregnancy negative test, normal liver function levels (ALT, AST), hemoglobin.

The 80 subjects will be divided into two groups of 40 each. The first group will be administered one dose of SEQ ID NO: 26 and a pharmacologically acceptable carrier per day. The second group will receive placebo once a day. Body weight and tumors (including tumor progression, arrest, degeneration, multiplicity) will be monitored every 3 days during the treatment period. The following tumor volume categories will be used for recording: Progression: When tumors grow at least 40% compared to the start of treatment; Suspension: The tumor has not changed more than 40% of its initial course throughout the course of treatment; Regression: Degeneration of more than 40% of the initial tumor area. Multiplicity: The appearance of new tumors during treatment.

Body weight : Differences in body weight will be observed between subjects receiving SEQ ID NO: 26 and pharmacologically acceptable carriers and those receiving placebo. Individuals receiving SEQ ID NO: 26 and pharmacologically acceptable carriers will weigh on average more than placebo subjects, given height and gender.

Tumor Volume: At the start of treatment, comparing tumor sizes in the two groups will not make much difference. On average, the tumor volume of placebo subjects will vary greatly. Individuals receiving SEQ ID NO: 26 and pharmacologically acceptable carriers will have reduced tumor size growth when compared to the placebo group.

Tumor Progression : As described above, the rate of tumor progression can be calculated. Individuals receiving SEQ ID NO: 26 and pharmacologically acceptable carriers will have less tumor progression in the course of the study than placebo individuals.

Tumor arrest : The rate at which the tumor stops as described above can be calculated. Individuals receiving SEQ ID NO: 26 and pharmacologically acceptable carriers will have more tumor arrest during the course of the study than placebo individuals.

Tumor Regression : As described above, the rate at which the tumor regresses can be calculated. Individuals receiving SEQ ID NO: 26 and pharmacologically acceptable carriers will have more tumor regression during the course of the study than placebo individuals.

Tumor Multiplicity : As above, the tumor can calculate the ratio of multiplicity. Individuals receiving SEQ ID NO: 26 and pharmacologically acceptable carriers will have less tumor multiplicity in the course of the study than placebo individuals.

Various modifications to the embodiments and systems described above will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with specific embodiments, the invention is not unduly limited to these specific embodiments. Indeed, various modifications of the above-described form for carrying out the invention which are obvious to those skilled in the relevant art are encompassed within the scope of the following claims.

                         SEQUENCE LISTING <110> Das Gupta, Tapas        Chakrabarty, Ananda   <120> COMPOSITIONS AND METHODS TO TREAT CANCER WITH CUPREDOXINS AND CPG         RICH DNA <130> 14PR-134419 <140> PCT / US07 / 086393 <141> 2007-12-04 <150> 60 / 872,471 <151> 2006-12-04 <160> 66 <170> PatentIn version 3.5 <210> 1 <211> 128 <212> PRT <213> Pseudomonas aeruginosa <400> 1 Ala Glu Cys Ser Val Asp Ile Gln Gly Asn Asp Gln Met Gln Phe Asn 1 5 10 15 Thr Asn Ala Ile Thr Val Asp Lys Ser Cys Lys Gln Phe Thr Val Asn             20 25 30 Leu Ser His Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met     50 55 60 Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val 65 70 75 80 Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr Met Phe Phe Cys             100 105 110 Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr Leu Thr Leu Lys         115 120 125 <210> 2 <211> 28 <212> PRT <213> Pseudomonas aeruginosa <400> 2 Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met Ala 1 5 10 15 Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp             20 25 <210> 3 <211> 105 <212> PRT <213> Phormidium laminosum <400> 3 Glu Thr Phe Thr Val Lys Met Gly Ala Asp Ser Gly Leu Leu Gln Phe 1 5 10 15 Glu Pro Ala Asn Val Thr Val His Pro Gly Asp Thr Val Lys Trp Val             20 25 30 Asn Asn Lys Leu Pro Pro His Asn Ile Leu Phe Asp Asp Lys Gln Val         35 40 45 Pro Gly Ala Ser Lys Glu Leu Ala Asp Lys Leu Ser His Ser Gln Leu     50 55 60 Met Phe Ser Pro Gly Glu Ser Tyr Glu Ile Thr Phe Ser Ser Asp Phe 65 70 75 80 Pro Ala Gly Thr Tyr Thr Tyr Tyr Cys Ala Pro His Arg Gly Ala Gly                 85 90 95 Met Val Gly Lys Ile Thr Val Glu Gly             100 105 <210> 4 <211> 155 <212> PRT <213> Thiobacillus ferrooxidans <400> 4 Gly Thr Leu Asp Thr Thr Trp Lys Glu Ala Thr Leu Pro Gln Val Lys 1 5 10 15 Ala Met Leu Glu Lys Asp Thr Gly Lys Val Ser Gly Asp Thr Val Thr             20 25 30 Tyr Ser Gly Lys Thr Val His Val Val Ala Ala Ala Val Leu Pro Gly         35 40 45 Phe Pro Phe Pro Ser Phe Glu Val His Asp Lys Lys Asn Pro Thr Leu     50 55 60 Glu Ile Pro Ala Gly Ala Thr Val Asp Val Thr Phe Ile Asn Thr Asn 65 70 75 80 Lys Gly Phe Gly His Ser Phe Asp Ile Thr Lys Lys Gly Pro Pro Tyr                 85 90 95 Ala Val Met Pro Val Ile Asp Pro Ile Val Ala Gly Thr Gly Phe Ser             100 105 110 Pro Val Pro Lys Asp Gly Lys Phe Gly Tyr Thr Asp Phe Thr Trp His         115 120 125 Pro Thr Ala Gly Thr Tyr Tyr Tyr Val Cys Gln Ile Pro Gly His Ala     130 135 140 Ala Thr Gly Met Phe Gly Lys Ile Val Val Lys 145 150 155 <210> 5 <211> 124 <212> PRT <213> Achromabacter cycloclastes <400> 5 Ala Asp Phe Glu Val His Met Leu Asn Lys Gly Lys Asp Gly Ala Met 1 5 10 15 Val Phe Glu Pro Ala Ser Leu Lys Val Ala Pro Gly Asp Thr Val Thr             20 25 30 Phe Ile Pro Thr Asp Lys Gly His Asn Val Glu Thr Ile Lys Gly Met         35 40 45 Ile Pro Asp Gly Ala Glu Ala Phe Lys Ser Lys Ile Asn Glu Asn Tyr     50 55 60 Lys Val Thr Phe Thr Ala Pro Gly Val Tyr Gly Val Lys Cys Thr Pro 65 70 75 80 His Tyr Gly Met Gly Met Val Gly Val Val Gln Val Gly Asp Ala Pro                 85 90 95 Ala Asn Leu Glu Ala Val Lys Gly Ala Lys Asn Pro Lys Lys Ala Gln             100 105 110 Glu Arg Leu Asp Ala Ala Leu Ala Ala Leu Gly Asn         115 120 <210> 6 <211> 128 <212> PRT <213> Alcaligenes faecalis <400> 6 Ala Cys Asp Val Ser Ile Glu Gly Asn Asp Ser Met Gln Phe Asn Thr 1 5 10 15 Lys Ser Ile Val Val Asp Lys Thr Cys Lys Glu Phe Thr Ile Asn Leu             20 25 30 Lys His Thr Gly Lys Leu Pro Lys Ala Ala Met Gly His Asn Val Val         35 40 45 Val Ser Lys Lys Ser Asp Glu Ser Ala Val Ala Thr Asp Gly Met Lys     50 55 60 Ala Gly Leu Asn Asn Asp Tyr Val Lys Ala Gly Asp Glu Arg Val Ile 65 70 75 80 Ala His Thr Ser Val Ile Gly Gly Gly Glu Thr Asp Ser Val Thr Phe                 85 90 95 Asp Val Ser Lys Leu Lys Glu Gly Glu Asp Tyr Ala Phe Phe Cys Ser             100 105 110 Phe Pro Gly His Trp Ser Ile Met Lys Gly Thr Ile Glu Leu Gly Ser         115 120 125 <210> 7 <211> 129 <212> PRT <213> Achromobacter xylosoxidans ssp. denitrificans I <400> 7 Ala Gln Cys Glu Ala Thr Ile Glu Ser Asn Asp Ala Met Gln Tyr Asn 1 5 10 15 Leu Lys Glu Met Val Val Asp Lys Ser Cys Lys Gln Phe Thr Val His             20 25 30 Leu Lys His Val Gly Lys Met Ala Lys Val Ala Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Glu Ala Asp Lys Gln Gly Val Ala Thr Asp Gly Met     50 55 60 Asn Ala Gly Leu Ala Gln Asp Tyr Val Lys Ala Gly Asp Thr Arg Val 65 70 75 80 Ile Ala His Thr Lys Val Ile Gly Gly Gly Glu Ser Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Thr Pro Gly Glu Ala Tyr Ala Tyr Phe Cys             100 105 110 Ser Phe Pro Gly His Trp Ala Met Met Lys Gly Thr Leu Lys Leu Ser         115 120 125 Asn      <210> 8 <211> 129 <212> PRT <213> Bordetella bronchiseptica <400> 8 Ala Glu Cys Ser Val Asp Ile Ala Gly Thr Asp Gln Met Gln Phe Asp 1 5 10 15 Lys Lys Ala Ile Glu Val Ser Lys Ser Cys Lys Gln Phe Thr Val Asn             20 25 30 Leu Lys His Thr Gly Lys Leu Pro Arg Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile     50 55 60 Ala Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp Thr Arg Val 65 70 75 80 Leu Ala His Thr Lys Val Leu Gly Gly Gly Glu Ser Asp Ser Val Thr                 85 90 95 Phe Asp Val Ala Lys Leu Ala Gly Asp Asp Tyr Thr Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Gly Ala Leu Met Lys Gly Thr Leu Lys Leu Val         115 120 125 Asp      <210> 9 <211> 129 <212> PRT <213> Methylomonas sp. J. <400> 9 Ala Ser Cys Glu Thr Thr Val Thr Ser Gly Asp Thr Met Thr Tyr Ser 1 5 10 15 Thr Arg Ser Ile Ser Val Pro Ala Ser Cys Ala Glu Phe Thr Val Asn             20 25 30 Phe Glu His Lys Gly His Met Pro Lys Thr Gly Met Gly His Asn Trp         35 40 45 Val Leu Ala Lys Ser Ala Asp Val Gly Asp Val Ala Lys Glu Gly Ala     50 55 60 His Ala Gly Ala Asp Asn Asn Phe Val Thr Pro Gly Asp Lys Arg Val 65 70 75 80 Ile Ala Phe Thr Pro Ile Ile Gly Gly Gly Glu Lys Thr Ser Val Lys                 85 90 95 Phe Lys Val Ser Ala Leu Ser Lys Asp Glu Ala Tyr Thr Tyr Phe Cys             100 105 110 Ser Tyr Pro Gly His Phe Ser Met Met Arg Gly Thr Leu Lys Leu Glu         115 120 125 Glu      <210> 10 <211> 166 <212> PRT <213> Neisseria meningitidis <400> 10 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Ala 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Thr Ser Met Gly His Asn Ile Val Ile Gly Lys Thr Glu Asp Met Asp                 85 90 95 Gly Ile Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ser Ser Le Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Glu     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 11 <211> 128 <212> PRT <213> Pseudomomas fluorescens <400> 11 Ala Glu Cys Lys Thr Thr Ile Asp Ser Thr Asp Gln Met Ser Phe Asn 1 5 10 15 Thr Lys Ala Ile Glu Ile Asp Lys Ala Cys Lys Thr Phe Thr Val Glu             20 25 30 Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Leu         35 40 45 Val Ile Ser Lys Gln Ala Asp Met Gln Pro Ile Ala Thr Asp Gly Leu     50 55 60 Ser Ala Gly Ile Asp Lys Asn Tyr Leu Lys Glu Gly Asp Thr Arg Val 65 70 75 80 Ile Ala His Thr Lys Val Ile Gly Ala Gly Glu Lys Asp Ser Leu Thr                 85 90 95 Ile Asp Val Ser Lys Leu Asn Ala Ala Glu Lys Tyr Gly Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Ile Ser Met Met Lys Gly Thr Val Thr Leu Lys         115 120 125 <210> 12 <211> 128 <212> PRT <213> Pseudomonas chlororaphis <400> 12 Ala Glu Cys Lys Val Asp Val Asp Ser Thr Asp Gln Met Ser Phe Asn 1 5 10 15 Thr Lys Glu Ile Thr Ile Asp Lys Ser Cys Lys Thr Phe Thr Val Asn             20 25 30 Leu Thr His Ser Gly Ser Leu Pro Lys Asn Val Met Gly His Asn Trp         35 40 45 Val Leu Ser Lys Ser Ala Asp Met Ala Gly Ile Ala Thr Asp Gly Met     50 55 60 Ala Ala Gly Ile Asp Lys Asp Tyr Leu Lys Pro Gly Asp Ser Arg Val 65 70 75 80 Ile Ala His Thr Lys Ile Ile Gly Ser Gly Glu Lys Asp Ser Val Thr                 85 90 95 Phe Asp Val Ser Lys Leu Thr Ala Gly Glu Ser Tyr Glu Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Asn Ser Met Met Lys Gly Ala Val Val Leu Lys         115 120 125 <210> 13 <211> 129 <212> PRT <213> Xylella fastidiosa 9a5c <400> 13 Lys Thr Cys Ala Val Thr Ile Ser Ala Asn Asp Gln Met Lys Phe Asp 1 5 10 15 Gln Asn Thr Ile Lys Ile Ala Ala Glu Cys Thr His Val Asn Leu Thr             20 25 30 Leu Thr His Thr Gly Lys Lys Ser Ala Arg Val Met Gly His Asn Trp         35 40 45 Val Leu Thr Lys Thr Thr Asp Met Gln Ala Val Ala Leu Ala Gly Leu     50 55 60 His Ala Thr Leu Ala Asp Asn Tyr Val Pro Lys Ala Asp Pro Arg Val 65 70 75 80 Ile Ala His Thr Ala Ile Ile Gly Gly Gly Glu Arg Thr Ser Ile Thr                 85 90 95 Phe Pro Thr Asn Thr Leu Ser Lys Asn Val Ser Tyr Thr Phe Phe Cys             100 105 110 Ser Phe Pro Gly His Trp Ala Leu Met Lys Gly Thr Leu Asn Phe Gly         115 120 125 Gly      <210> 14 <211> 138 <212> PRT Cucumis sativus <400> 14 Met Gln Ser Thr Val His Ile Val Gly Asp Asn Thr Gly Trp Ser Val 1 5 10 15 Pro Ser Ser Pro Asn Phe Tyr Ser Gln Trp Ala Ala Gly Lys Thr Phe             20 25 30 Arg Val Gly Asp Ser Leu Gln Phe Asn Phe Pro Ala Asn Ala His Asn         35 40 45 Val His Glu Met Glu Thr Lys Gln Ser Phe Asp Ala Cys Asn Phe Val     50 55 60 Asn Ser Asp Asn Asp Val Glu Arg Thr Ser Pro Val Ile Glu Arg Leu 65 70 75 80 Asp Glu Leu Gly Met His Tyr Phe Val Cys Thr Val Gly Thr His Cys                 85 90 95 Ser Asn Gly Gln Lys Leu Ser Ile Asn Val Val Ala Ala Asn Ala Thr             100 105 110 Val Ser Met Pro Pro Pro Ser Ser Pro Pro Ser Ser Val Met Pro         115 120 125 Pro Pro Val Met Pro Pro Pro Ser Pro Ser     130 135 <210> 15 <211> 162 <212> PRT <213> Chloroflexus aurantiacus <400> 15 Met Lys Ile Thr Leu Arg Met Met Val Leu Ala Val Leu Thr Ala Met 1 5 10 15 Ala Met Val Leu Ala Ala Cys Gly Gly Gly Gly Ser Ser Gly Gly Ser             20 25 30 Thr Gly Gly Gly Ser Gly Ser Gly Pro Val Thr Ile Glu Ile Gly Ser         35 40 45 Lys Gly Glu Glu Leu Ala Phe Asp Lys Thr Glu Leu Thr Val Ser Ala     50 55 60 Gly Gln Thr Val Thr Ile Arg Phe Lys Asn Asn Ser Ala Val Gln Gln 65 70 75 80 His Asn Trp Ile Leu Val Lys Gly Gly Glu Ala Glu Ala Ala Asn Ile                 85 90 95 Ala Asn Ala Gly Leu Ser Ala Gly Pro Ala Ala Asn Tyr Leu Pro Ala             100 105 110 Asp Lys Ser Asn Ile Ile Ala Glu Ser Pro Leu Ala Asn Gly Asn Glu         115 120 125 Thr Val Glu Val Thr Phe Thr Ala Pro Ala Ala Gly Thr Tyr Leu Tyr     130 135 140 Ile Cys Thr Val Pro Gly His Tyr Pro Leu Met Gln Gly Lys Leu Val 145 150 155 160 Val asn          <210> 16 <211> 140 <212> PRT <213> Chloroflexus aurantiacus <400> 16 Ala Ala Asn Ala Pro Gly Gly Ser Asn Val Val Asn Glu Thr Pro Ala 1 5 10 15 Gln Thr Val Glu Val Arg Ala Ala Pro Asp Ala Leu Ala Phe Ala Gln             20 25 30 Thr Ser Leu Ser Leu Pro Ala Asn Thr Val Val Arg Leu Asp Phe Val         35 40 45 Asn Gln Asn Asn Leu Gly Val Gln His Asn Trp Val Leu Val Asn Gly     50 55 60 Gly Asp Asp Val Ala Ala Ala Val Asn Thr Ala Ala Gln Asn Asn Ala 65 70 75 80 Asp Ala Leu Phe Val Pro Pro Pro Asp Thr Pro Asn Ala Leu Ala Trp                 85 90 95 Thr Ala Met Leu Asn Ala Gly Glu Ser Gly Ser Val Thr Phe Arg Thr             100 105 110 Pro Ala Pro Gly Thr Tyr Leu Tyr Ile Cys Thr Phe Pro Gly His Tyr         115 120 125 Leu Ala Gly Met Lys Gly Thr Leu Thr Val Thr Pro     130 135 140 <210> 17 <211> 96 <212> PRT Cucumis sativus <400> 17 Ala Val Tyr Val Val Gly Gly Ser Gly Gly Trp Thr Phe Asn Thr Glu 1 5 10 15 Ser Trp Pro Lys Gly Lys Arg Phe Arg Ala Gly Asp Ile Leu Leu Phe             20 25 30 Asn Tyr Asn Pro Ser Met His Asn Val Val Val Val Asn Gln Gly Gly         35 40 45 Phe Ser Thr Cys Asn Thr Pro Ala Gly Ala Lys Val Tyr Thr Ser Gly     50 55 60 Arg Asp Gln Ile Lys Leu Pro Lys Gly Gln Ser Tyr Phe Ile Cys Asn 65 70 75 80 Phe Pro Gly His Cys Gln Ser Gly Met Lys Ile Ala Val Asn Ala Leu                 85 90 95 <210> 18 <211> 166 <212> PRT <213> Neisseria gonorrhoeae F62 <400> 18 Cys Ser Gln Glu Pro Ala Ala Pro Ala Ala Glu Ala Thr Pro Ala Gly 1 5 10 15 Glu Ala Pro Ala Ser Glu Ala Pro Ala Ala Glu Ala Ala Pro Ala Asp             20 25 30 Ala Ala Glu Ala Pro Ala Ala Gly Asn Cys Ala Ala Thr Val Glu Ser         35 40 45 Asn Asp Asn Met Gln Phe Asn Thr Lys Asp Ile Gln Val Ser Lys Ala     50 55 60 Cys Lys Glu Phe Thr Ile Thr Leu Lys His Thr Gly Thr Gln Pro Lys 65 70 75 80 Ala Ser Met Gly His Asn Leu Val Ile Ala Lys Ala Glu Asp Met Asp                 85 90 95 Gly Val Phe Lys Asp Gly Val Gly Ala Ala Asp Thr Asp Tyr Val Lys             100 105 110 Pro Asp Asp Ala Arg Val Val Ala His Thr Lys Leu Ile Gly Gly Gly         115 120 125 Glu Glu Ser Ser Leu Thr Leu Asp Pro Ala Lys Leu Ala Asp Gly Asp     130 135 140 Tyr Lys Phe Ala Cys Thr Phe Pro Gly His Gly Ala Leu Met Asn Gly 145 150 155 160 Lys Val Thr Leu Val Asp                 165 <210> 19 <211> 150 <212> PRT <213> Vibrio parahaemolyticus <400> 19 Met Ser Leu Arg Ile Leu Ala Ala Thr Leu Ala Leu Ala Gly Leu Ser 1 5 10 15 Phe Gly Ala Gln Ala Ser Ala Glu Cys Glu Val Ser Ile Asp Ala Asn             20 25 30 Asp Met Met Gln Phe Ser Thr Lys Thr Leu Ser Val Pro Ala Thr Cys         35 40 45 Lys Glu Val Thr Leu Thr Leu Asn His Thr Gly Lys Met Pro Ala Gln     50 55 60 Ser Met Gly His Asn Val Val Ile Ala Asp Thr Ala Asn Ile Gln Ala 65 70 75 80 Val Gly Thr Asp Gly Met Ser Ala Gly Ala Asp Asn Ser Tyr Val Lys                 85 90 95 Pro Asp Asp Glu Arg Val Tyr Ala His Thr Lys Val Val Gly Gly Gly             100 105 110 Glu Ser Thr Ser Ile Thr Phe Ser Thr Glu Lys Met Thr Ala Gly Gly         115 120 125 Asp Tyr Ser Phe Phe Cys Ser Phe Pro Gly His Trp Ala Ile Met Gln     130 135 140 Gly Lys Phe Glu Phe Lys 145 150 <210> 20 <211> 33 <212> PRT <213> Chloroflexus aurantiacus <400> 20 His Asn Trp Val Leu Val Asn Gly Gly Asp Asp Val Ala Ala Ala Val 1 5 10 15 Asn Thr Ala Ala Gln Asn Asn Ala Asp Ala Leu Phe Val Pro Pro Pro             20 25 30 Asp      <210> 21 <211> 28 <212> PRT <213> Bordetella pertussis <400> 21 Leu Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile Ala 1 5 10 15 Ala Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp             20 25 <210> 22 <211> 27 <212> PRT <213> Neisseria meningitidis <400> 22 Ile Gly Lys Thr Glu Asp Met Asp Gly Ile Phe Lys Asp Gly Val Gly 1 5 10 15 Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp             20 25 <210> 23 <211> 27 <212> PRT <213> Pseudomonas syringae <400> 23 Ser Lys Lys Ala Asp Ala Ser Ala Ile Thr Thr Asp Gly Met Ser Val 1 5 10 15 Gly Ile Asp Lys Asp Tyr Val Lys Pro Asp Asp             20 25 <210> 24 <211> 27 <212> PRT <213> Vibrio parahaemolyticus <400> 24 Ala Asp Thr Ala Asn Ile Gln Ala Val Gly Thr Asp Gly Met Ser Ala 1 5 10 15 Gly Ala Asp Asn Ser Tyr Val Lys Pro Asp Asp             20 25 <210> 25 <211> 27 <212> PRT <213> Bordetella bronchiseptica <400> 25 Thr Lys Thr Ala Asp Met Gln Ala Val Glu Lys Asp Gly Ile Ala Ala 1 5 10 15 Gly Leu Asp Asn Gln Tyr Leu Lys Ala Gly Asp             20 25 <210> 26 <211> 592 <212> DNA <213> Pseudomonas aeruginosa <400> 26 cgtggcaggc atacagcatt tcaatcggtt cggcaaaggt aacgctttgg gtttcaagcg 60 catgtgcctg tttcgctact ccaccggcac gtttaaaggc ggacgttcta taatagaaca 120 tcctgcacaa agcacgctgg ttgtcccttt cacacattta gcagaactac ggcaactgcc 180 tcacttggct gcttgtcaaa gcaccgtgac ccgggaaggt gcacgcaaat ttgtagtcgc 240 cgtcagccaa tttggcagga tccaaagtca gggaagactc ttcgccgccg ccgatcagtt 300 tggtgtgggc aacaacgcgc gcatcatcag gtttgacata gtcagtatcg gcagcaccta 360 cgccgtcttt aaatacgccg tccaagactt cagctcaggc aaacacaatg ttgtgaccaa 420 tgctggcttt gggttgcata ccaaaatgat taagatatat aaaaaaatca taaaaaaatt 480 aattagtaaa ataattatat tataaaacta catatagagc gctaagaaaa cattggataa 540 aaagaaagag atcatttgtc gaatatatat atataataaa atgtatttta at 592 <210> 27 <211> 612 <212> DNA <213> Pseudomonas aeruginosa <400> 27 atacctgccc ggtacttctg gaacagcgtc gtcaccttat cgggagatac cgagtaggtg 60 atgccgacgg cgccgccgac cttcatgccc tcaacggtct ggaagctgat cgcttcctcg 120 ccgccccagg tctcggtctg cgtgaaggtg gggaacaggt agagctcctc gttcacgcct 180 acccagtagc gcccagttcc gacctcacgc gtctccacac ccttctcgga gccgtagaga 240 ttgacgatca cgccgacgtt gccggcaggc accttcgaac agcccgccag gacggcgagc 300 aggcacagca ttgcagcagc gggaatccgc ttcattggtc tttctccttg ctggtggtgg 360 ccgcttgttc gcggcgggtg ttggcgaggt ggacgccgag gcagaccgag gcgatcaacc 420 agacgccagg gatggcgaat cccgcgaaaa ccagaacgtc gtcgcgactg ctgaccaggg 480 ccggcccaat gccgcccacc agggcgactg acagtccggc ataggccagc agcgcgatac 540 agatcaggaa gagcttcccg ggcttgatga gaggtttctt gtccatgctt tcctccaggc 600 aagccgatgg cc 612 <210> 28 <211> 231 <212> DNA <213> Pseudomonas aeruginosa <400> 28 atatcccccg tatttccctc atatttggga tatcgtcgcg tttcaccgct ctcttagtta 60 ctcgcccccc ctctcccgcc tccccctcct cccctctccc tccctccgac ttccctgcgc 120 gcatcccgca tcccctctcc cctttcggat tgcccccttc accaacgcgt ccgtccccac 180 cgcttttctc gcccgctcag cgggccctac gcgcccccct cctccccgca t 231 <210> 29 <211> 782 <212> DNA <213> Pseudomonas aeruginosa <400> 29 tgagaccggg tacgagcttc ccaactggaa ccccgttcgc tgggaactgc gccacctgct 60 gatcgccctg cgcaccctcg cccccgcccc ggacagcccg ctgcacgccg gctacaacgg 120 catctcgccg tacaagctgg gcgaacacaa catcaagttc cgcgtcgtcc ccgccccgga 180 gaagtgcccg gcctaccagc taccgaagca gaaccaggac ctgcccaact tcctccgcgc 240 cgccctgtac cagcaactct ccatcgaccg cacccccgcc tgctacgcct tccaggtgca 300 gcgccaggac ccggccaagt acatgcccat cgaagacacc agcgtcgaat ggaaggagtc 360 ggacgcgccc ttcgctacca tagccgacat cattgtgcca gctcaggatt tcgatagccg 420 ggaacagaac ctgttctgtg acaacctttc gttcaacccc tggcacgcgc tgccggagca 480 tcggccgatc ggcgggatca accggttgcg gaaggcggtt tacgaggcgg tcagtgggta 540 taggttgggg aggaatgggt gagggtttag ggggaggagt gcttggtgtt acgttgatcg 600 ggaatggcca gaatcggcca gaaacggcca ttcgatcaac gcgctttcag gtctatcaag 660 cacgtggcgc gtcaccgttg agcaccttaa ctagaacgcg ttcctaccga ttgatacacg 720 accatcaact tacagtgtct ggcaggtaac aaagactcga atcgccctaa ggagtcgttc 780 at 782 <210> 30 <211> 115 <212> DNA <213> Pseudomonas aeruginosa <400> 30 agttcacgca ggaaccggtc gagcggtacg gcagcatcga tgcggggttc cgggatcccg 60 ccggcaatgg ctggaagatg atccagtcgc ccgcaggcgc ctcctgacga atagg 115 <210> 31 <211> 688 <212> DNA <213> Pseudomonas aeruginosa <400> 31 tagtcccggg tacgagaacc gcgaaggacc ggttcacctc cttctccttg gtgatgatgc 60 cagtgatcgc tccagacgta atgccgccaa tacgcacggc cgccccctgc cgtaccgtca 120 cggtcgtcgt ggattgtcct tcagggtagt cgtagggttc gcgaacgcgg cacagcgcgg 180 cgctattgtt gctaagcgcc acaatccaaa tctgatgctg atcttggtaa tcaagtaggt 240 tctccccgtt gtcgtcaaac cactcataac tcaccacggt attgcagacg tgcaggcctg 300 gagggaatgt tgccggcgca tcaagcttta cgccgcgata tcgatcggcc tgcaagaaac 360 tcgtcacatc atccgattcg ccagtgcatt gaatcgttcg agtcacagag aacccagtat 420 cgggcgaggc ctgcgcctcc aatatctcct tcagctcaat atgatctgca acttgtccag 480 agtccgttat gagttcaaga acgctggcgc gctccattct gctgtgctca acatcctgct 540 catatcggta agtcctcgta ccataatgtt gacggttata tctagccgac cgaacatttc 600 cctgtgcgtc ataccacgct gtgatcaatg ccgaggtctg agtccactcg tcacgaagat 660 cgccgtcaca acagggctcg tacccggg 688 <210> 32 <211> 777 <212> DNA <213> Pseudomonas aeruginosa <220> <221> misc_feature (222) (98) .. (98) N is a, c, g, or t <400> 32 atcagtcgct atcctatgta ttttggatgg cgtcttccct tttatagaga cggattgtaa 60 tatgaagtgc gacagctagg aaaaagaaaa ggcccatngc tggcatcgtg tacaatggaa 120 gttaccatac taaccatttt gtacaggagg acccaacatg agctactccc atcttagcat 180 aatcgagcgt ggacaactag aaactttgca tcgactcggt tggtcatgcc gggctatcgg 240 acttgaacta ggccgtcatc cttctaccat cgctcgagaa ttaaagcgag gcagcgacaa 300 tgagggctac tccgctgaat ccgctcagca agcgtcttac gagcgaagaa cgacatgcgt 360 gcctgctgga aagtacacac ccgagcttgc cgatgaaatc aacctgaagc taaaggaaac 420 ctggtcaccc gagcagatcg cggaaaaaag acgggcgaca ggggcgtttt tcgtatgctt 480 caaaacaatc tacagatggc tctactccgg gcgccttgca gccggagagg ttacggttct 540 acggcacaag gggaagcggc agaagccggt ggaaacacgc ggccgattcc gggtgggcac 600 cccgattagc aaacgtccga aagaagtccg cacacgcacg tcattcggcc attgggaact 660 cgatacggtt gtctccagcc gagggaaaag ccgggcttgt gccgccacct ttatggaaag 720 gaagacgcgc ctgtacatgg ctggaaaatg ccggctcgat tcgccctata ggagtcg 777 <210> 33 <211> 44 <212> DNA <213> Pseudomonas aeruginosa <400> 33 actggtcggt gcggcgttcg cctggagcgt cctggccgtg ccgg 44 <210> 34 <211> 603 <212> DNA <213> Pseudomonas aeruginosa <400> 34 atgatcgact ccttagggcg aatcgagtga attatcactg cggctttaaa aagttggcca 60 atttgctaag gatactggca gaatcattgc gtcatggaat tcactgatcg cctacgctga 120 atacattagt gatgccccaa aattggtggt ctgacgagaa cggctccaga tggtgggtca 180 tcaactttcg gaatacttag tcctttatat tagcgatcca aagttttatt aggtgaaatc 240 gaatgtcata tcacggagta gtgccacaat atcgctggat ttaatcgctt tggaagatat 300 ggatcatcta atgatagcaa tgtcgccaag tgtggttggt aaaatcattt cgtaatggag 360 ttcacgtaac gccttcgctc caaaacattg gtgataatgc aatatcaagt cgttggaatt 420 gcaataattc atcagttaca ttcggcaaac ccgcaacatc aacacaatat gtagaatagc 480 atttcaattt tgcgaagaag gcaagtcgct tcattacttt ataacactat gatttaatta 540 cttaacgcac taaagtgatg taactctcgc tctcggtatt tgcagtaatc ctcaagttgc 600 tta 603 <210> 35 <211> 621 <212> DNA <213> Pseudomonas aeruginosa <400> 35 tgatcgactc cttagggcga atcgagaagc ttacagttaa caaattttta cttttcccta 60 ttccatcagg tttatgcacc gaaaaagtgg tgatactggt ggatgaggtg ataaaaaatg 120 gaagatcaaa aaaatccaaa tcaaccgatt cctttgaaga agtcaaaatc ctggaaaacc 180 tttctgggga agaagtgggc gtttcccgca atctacatcg gcttagcagc aatcatcctc 240 gcattcgtga tgtggtatca gggcaacgtg tttcatgcag taagcgatga gcttagcaag 300 cagccgacgc cagtcgcaca gaaccaaccg gaaacaacgg caccaaacac agaagtttcg 360 caagatgatg cagtaccagt cagcaaggca acacaaccgc tcgcatggcc agtggcagca 420 agcgtgagct actccaaagc catggatttc ttcaatgatg cagctgcgaa agaagagcaa 480 gccaaagcgt tggtcaagta caacaactcg tacatcccgc acacagggat cgacattgtc 540 tccacggata aaaaaggatt tgatgtcgcc gctgccctcg atggcaaggt gaaaaagtgg 600 aaaatgatcc gttggtaggc a 621 <210> 36 <211> 876 <212> DNA <213> Pseudomonas aeruginosa <400> 36 ataagtacct ggtacgagca cttactgacg tactttctgc ggcagtacac aatacaatct 60 ttcggtagct caactggtcg agattgctgt taagcgggga gaaggtgtgc tcacagacaa 120 gggtgcactt aacgcgttga caggcaagtt caccggtcgt tccccgaaag acaaattcgt 180 tgtggacgaa gcatccgttc atgacaaaat caactgggga cctgtgaacc aaccgatttc 240 ccgtgaaaaa ttcgacattc tctacgcgga tgtgatggag catctgcaag gcaaagatct 300 gtttgttttc gacggttttg ccggtgcgga gaagacattc cgtctgccga tccgtgtagt 360 aaatgaatat gcatggcaca acctgtttgc tcgccaattg ttcgttcgcc catccgaggc 420 ggaattggct gatcataaag cggaattcac ccgtcgtata tgcacctagc tacaaggcga 480 atcctgcggt tcacggcacc gactccgaga cgttcatgct tatgagcttt gggcaaagag 540 tggtgctgat ctgcggaacc gaatacgccg gcgaaaggta agaatcgatc tgcagcgtga 600 tgggctggct cccgccttgc acaaaatgtt ttgtcgatgc cctgctcggc aaacgttgcg 660 cagcgaagta gatgtccctc tgatcttcgg tcttgccggc acaggcagat ggacgctatc 720 ggggtgagcc cggtcatgag cagattcggg ggaagcaatt ccggatggtc ggtaagacgt 780 gcgtatttga ggggagcgag tgggggccgc ggcgagacgg tgcgcgactg tgggaaaagg 840 taatccccaa tatatggcgc ctcttatcct cttaat 876 <210> 37 <211> 47 <212> DNA <213> Pseudomonas aeruginosa <400> 37 cacccagctc gatacggtga gcatgtacat gtcccgcggg cgcaggt 47 <210> 38 <211> 631 <212> DNA <213> Pseudomonas aeruginosa <400> 38 tatcgactcc tatagggcga atcgaggctc gaattaccct ctgacggggg cacctccggg 60 aggacccgcc aaattttcaa acttgtgctg gacaacaaga attcgcacca ctttggtgca 120 ctcttcagcg cctcgcggcg aaccgagcgg caacgccgcg catcgccacc tcgaactcac 180 gcggcaggtt ctcgtcggtg tactgctgcg cgatctcgaa gaagctcagc cggcggcgat 240 acgaagggcg tgacacgaag gccatgatga tcgagacagc atcccggcct cggcctgttc 300 gctcagcaat gccaatgggc tggcccttgc gggtcatgac gaagtagcgg cgagcattac 360 ccttcgccct gctccgtctg ctatcggtcg cgttcgcgtt gtacccggcc tgagtgaagc 420 cccgaatacc gctcagcgct ctggtcactt gacctcgcct gatgttcccg tagcgatcaa 480 gatcagcacc ggcgccgggc accacgtact taccttcggg caggatcccc ttggccctga 540 gctgaagctc ggccggcttg ttccgacgcg gcccaccgta gacctcgggg gcaatccaca 600 ccgatgcagg ctgcgcaccg tccgcttcgt a 631 <210> 39 <211> 148 <212> DNA <213> Pseudomonas aeruginosa <400> 39 gatattgctc gacaggtagg tgcggtgcac gccgatcacc ggatcgcccc agttgaagac 60 caggtcggtg gtcatgtcga aatcatggct ggcgatgcgc ttggcccagg tcgggaagtc 120 gggcgaagcg cgcacctgca ccgcgatc 148 <210> 40 <211> 619 <212> DNA <213> Pseudomonas aeruginosa <400> 40 tgagacccgg gtacgagcag cggcagttcg cgcagaccac gcgccagtgc gatggacggc 60 gggtcgccgg cgcgtccgcc tgagtagttc tccaggccga tgtggataag tccgccgagg 120 aaggtgccgg tagtcaggac cacgttgtcg gcatggaagc gcagtcccat ctgggtcact 180 acgccgcgga cctgatcctg ctcgacgatc aggtcgtcgc aggcctgctg gaatatccac 240 aggttcggct ggttttccag tgtatggcgg atagcggcct tatagagcac gcgatcggct 300 tgtgcacgag tggcgcgcac tgccggcccc ttgcggctgt taaggatgcg gaactggata 360 ccgcccttgt cggtggcttc ggccatggcg ccgccaaggg cgtcgatttc cttgaccaga 420 tggctcttgc cgatgccgcc gatggcgggg ttgcagctca tctgccccag ggtttccaca 480 ttgtgggtca acagcagggt tttcacaccc atgcgcgcag ccgcgagtgc ggcttcggtg 540 cctgcatggc caccgccgat cacgattacg tcaaaacggg tagggaaatc caccacgcac 600 ctcggcctgt tcagaagca 619 <210> 41 <211> 587 <212> DNA <213> Pseudomonas aeruginosa <400> 41 actacctact gctactgacc ttttatccat cgttgcctct aagtttctgt accgagatat 60 atctccggtg agcagtaact caagagcatc aaaaaatgag gttttcccaa agccattcgg 120 tccatcaagg acagttaggt tctttgagcc aatattgaaa acctgaaatc gaaatgcctt 180 aaaattcctc agagcaacct tatttataat taacttcata acctgcctcg gcgatagcat 240 ctaccaaatc agccagtgtc tttaccgggc tatctgttag atccatattt agaacgaagg 300 actctataaa tgaaagaagt tccggctgcc ttgtaccaag aactctttgg gtatgttttg 360 agtgcagtcc ctcaatgtcg actggagtac cgtctccaat atcaataaag ctaagtttta 420 taacaattct atagagcagt tcattccaac tctccaaccc aatcatctcc ttatagcgag 480 agaaggtgtc aggatcattt atcagaacac tcaatatatc gctcaggctc tcaaaaccaa 540 ttctcgcctt cagattatca agctcagagg gggtgtaata tagaaca 587 <210> 42 <211> 190 <212> DNA <213> Pseudomonas aeruginosa <400> 42 cggccagcgc ctgaaggatc tctgctccag gggcgtgatg tggatcgccg agacctgggc 60 ggagatcgac aagtggatct tcgccacctg cacgccgacg gtctgggacg tgcgcggcgc 120 cggcgacctg cgccaggaca cctcgtacct ggtggtgagc aaccaccagt cctgggtcga 180 catccccgcc 190 <210> 43 <211> 129 <212> DNA <213> Pseudomonas aeruginosa <400> 43 agcttagaac ctttaccaaa ggtgatgcgg agagatgggt aagcacaacc aaaaaagcca 60 gtgattctgc attctggctt gaggttgaag gtaattccat gaccgcacca acaggctcca 120 agccaagct 129 <210> 44 <211> 619 <212> DNA <213> Pseudomonas aeruginosa <400> 44 tgagacccgg gtaccgaggg taattcgagc cccgctcgcc aaatatgtat gaccattttt 60 cggaggttgg ttgttgttta gtcatgagca aaaacgaaac aaccaaacag cgcggatggt 120 tgaacaagtc cgagatggcc gcgagcctcg ggatttctcc gcaagccttt gataaatggg 180 gcgttcaacc aatcgagcga ataggtcgag aggccttcta cacggtggcg gatgtggtcg 240 aaaaccgcat ccagcacgcc gctcggaaac aacaacctga gggggagcta ccggaaggtc 300 tcgatcccta cgctgaagcc aagctgacac aggagcgact ccggctcacc aaggcccagg 360 cctacgccca agagcagaag aaccaggtcc aggacaagct cctggtcccg gtcccgttcg 420 ccactttcgc cttggcgaag atcgccgcca agattggctc ggcgctggag accgtctgca 480 aaacggtcag tcgccgccac ccggatgctg atcccttgct gatggagtcc ttcgagcggg 540 agatcgcctt ggcgcgaaac ctttccgctg agttcagcga cgacatcccg ggaatccttg 600 atgagtacct tgcaaccct 619 <210> 45 <211> 912 <212> DNA <213> Pseudomonas aeruginosa <400> 45 atacaataaa cgtcgagacg tttattgctt taacctttgg agaatcactc tccgcctggc 60 aacgtcctac tctcccagtc cccttcggga caagtaccat cggcgctgga gggcttaacg 120 gccgtgttcg gtatgggaac gggtgtgtcc cctccgccat catcaccaga cgatgcacat 180 ggatgtgcta gtgtcagcgt tgcgacagga tgtcgcgcac ttagctgacc ttattgattc 240 tttttgctaa aagcgacagg cactaatgta tcacatctgc accatcgcaa tcaagcactt 300 ttttaagaaa aatggtggag ctgaacggga tcgaaccgat gacctcctgc ttgcaaggca 360 ggcgctctcc caactgagct acagccccat aatgggtata tgaaggcgaa aatggtgggc 420 ctaggctgac tcgaacagcc gacctcacgc ttatcaggcg tgcgctctaa ccaactgagc 480 tataggcccg agtttcgggt gcccttacat gctccctcaa aactgaacag cgagcgaaag 540 atctccatag aaaggaggtg atccatccgc accttccggt acggatacct tgttacgact 600 tcaccccagt catctacccc accttcggcg gctggctcct tacggttacc tcaccgactt 660 cgggggttgc aaactcccgt ggtgtgacgg gcggtgtgta caaggcccgg gaacgtattc 720 accgcggcat gctgatccgc gattactagc gattccgact tcatgcaggc gagttgcagg 780 ctgcgatccg aactgagact ggttttaaga gatttgcgaa gtctcgggag cgaacatccc 840 ggtgcaccag gcattggagc acgtgtggac gcccgggcct aggggggatg atggtttggc 900 ctcgaccccg gc 912 <210> 46 <211> 269 <212> DNA <213> Pseudomonas aeruginosa <400> 46 gggacagctg accggtgaac tgggtgccgt gcagaaccgt ctggaatcga ccatcgccaa 60 cctgaacaac gtggttaaca acctgtcgaa cgcccgttcg cgcatccagg acgccgacta 120 cgccacggaa gtgtcgaaca tgtcgaaggc ccagatcctg caacaggctg gcacctcggt 180 tctggcgcaa gccaaccaag tgccgcaaac cgttctgtcg ctgctgcgtt aattcacggc 240 aaagcagtac ggacggggga agctccggc 269 <210> 47 <211> 608 <212> DNA <213> Pseudomonas aeruginosa <400> 47 tgagatccgg gtacgaggtc acgaccgaaa tgttctccct gttgactgcc ttgttgtact 60 ggttttccgt tttggtcagc tcttcctccg cctgcacaac atccagagaa gtggacaacc 120 cgttctcata gcgaatctgt gctgcgcgga aattttcttc cgccatagcc tgcgactcct 180 tgtacatgtc gacgctggac agggatgctt ccatattgaa atacgcctgt tttacttcca 240 cttcaatgct gcgtttcgta tcctccagat cgattttcgt cgcctccaga tcattttttg 300 ccatggagcc ttgataggtc gacaaggccg aatacttttg gaacagctcg acattcaatt 360 cggcgagctg gatctcgttt tgcttttgct gaatttcgta acgcttttcg ccagcttgct 420 tcaatgcctc atccaggctg ccaattgtcg gcttcgtcag ctgcgtttcc ttgaccaacg 480 tccacttttt gtccaaatcg acacccaggt agttgttcaa gttcaagaat gcgaccggta 540 ccgcattttg cgcgctgagc aaagatgcct tcgcattcat cacgctcacg ctgtgcggac 600 ggcaagtc 608 <210> 48 <211> 119 <212> DNA <213> Pseudomonas aeruginosa <400> 48 cgtttcatgc gcgttatttc gcgattaata tcggcggtgc gatcgggccg cttgttggct 60 tgaagctcgg cgccggaggg tccgcctcgt ttttgccgtt tttggtcagc agcgcgatc 119 <210> 49 <211> 305 <212> DNA <213> Pseudomonas aeruginosa <400> 49 ctcccccccc ccgcgaccgc cctccccgcc gcccgggtgc atgctgcggt cgcttcgccg 60 cgctccccgc gccccccgcc cccccctccg tccccgcccc cgcgctcccc tctcctcact 120 ctccccccgc tccctccgcg caccctgttc gcccccgcgc cccccgcccc ccccgccccc 180 cccatctctc ttcccacccc gccgcctcac gtttcttaac ccgtcagcgg ctcactgacc 240 cccacccgac cccacctcac tacctccccc cctcaggccc cccacgcctt ccccccccat 300 ccttc 305 <210> 50 <211> 71 <212> DNA <213> Pseudomonas aeruginosa <400> 50 caattctaat atgagaatgg ttttcattaa aaattgagag cgggcggcgc cgcgcacggg 60 gcgcggtagt c 71 <210> 51 <211> 136 <212> DNA <213> Pseudomonas aeruginosa <400> 51 atcgtggagc ctgtgatggg ggctgccggc gtcattcctc ctttgccggg atatttggag 60 ggattgcgcg agcttgcgaa ccgctatgac gtccttttga tttttgacga ggtgcagaca 120 ttgcgcttaa gcacag 136 <210> 52 <211> 49 <212> DNA <213> Pseudomonas aeruginosa <400> 52 tactcaatgg cggaaatggc gcatgccagt gaacaccatc gcgatatcc 49 <210> 53 <211> 792 <212> DNA <213> Pseudomonas aeruginosa <400> 53 atctcgtccg cctgctgcga agtgccatcg atggcgcgct cggcccactc ggggagctgc 60 ctttgcaacc gcatttcgtt gaggatcgtc cagaggcgcg aggtcaggtc gcgctctgcc 120 cggatcccgt ggcggagcat ggtgattgag gagttcattg ctggtcctcc tcacgcaggg 180 agtcgagcgc ggcgtcaacc aaatcgccag ccatctctgc agcaatctcc aaggcataca 240 agcacgcgtg ctcttcgtcg gaggtggtca gtgctccgag aatgctagaa acacttagcg 300 tcagggcgat ggccgtgctc aaggcctctt caaccgtcgt ggtcgggtta atcgctgcga 360 atcttcgcgg cggaagctga gacaccggcg ccttcagtac ggccggcccg agcttgagcg 420 cgctcatgcc gcaccgcctt cgtgtcgcga cacgttttca gcatttctgg attgggtcgc 480 gacaccagcc cggcaagctc tgagcaatgc gccagccatg ctccccagca gggcgagagt 540 attgagttcc tgagagagca gcggctcgcc ggcatcgtcc atcgcccggg tcattctcaa 600 aataatcagg ggaaccgcct cggtgatgtg ctcggggggg tgccgagtcg cgtcaacctg 660 gcgggccgta acactgtaga acaacaactc gtccccggtg agcggatctc aggaaatgtc 720 ggggggcgag ggggcgggcg cctggggaga ggagggcgtg ggtgatgtgg gctcgggggg 780 gcgagggggg ag 792 <210> 54 <211> 229 <212> DNA <213> Pseudomonas aeruginosa <400> 54 ttcgtcgtgc acgtagacct tctgcacctg tacctggttg ctcatcagga ccaggtcgtt 60 ggcgccgacg atctcgtagt tgatcgtgtt ggtcagctcg aactccgcgc cgcgcgcgga 120 accggtgtag ctgacggtgc gctgctggtt gtcctcgcgg accagcacca ggtggtaggg 180 cgcgttgctg gtgaccttca cgccgctgtt ttccagggtt tccttcagt 229 <210> 55 <211> 612 <212> DNA <213> Pseudomonas aeruginosa <400> 55 atgatcgact ccttagggcg aatcgagcaa ctactacagc ccacagcctc gttatccgag 60 gcaactacgt ataactgctc ccgagatacc cgcctcgctc cgcgatgaat attcgccgga 120 gataggcgcg tccttccgcg tctcggttcc tgatgatttg ctggagatgt agccatgggc 180 caacgctgga tcaacaactg gcagacagag ctctcgggtc cgctatcggc gggtggggtt 240 tctcttacca tccccgctgc cgcggctgat cttctgccca tctcggctcc ttctgatttc 300 atcctgctca ctcttgccga tgagtcaggc tctgttcacg aagtggtcaa ggcgacggca 360 aagagcggcg ggaatatcac tgttgcgaga gcctctgagg gtgtgcccgt tgagtggcca 420 tcgggctcga aagtgtacgc ggcggcgacg gcgggaactc tcgcaagcat cgagaaccga 480 atcaccatcc tcgagcaggc tggtcctggg cctggtggcg gcacgttcaa ggcgcaggag 540 gtcaacgact cgactccggt agccctcgcc tcggatacaa cgatcgtgcg cgtatccgca 600 tcgtttgatg gg 612 <210> 56 <211> 593 <212> DNA <213> Pseudomonas aeruginosa <400> 56 atctaccttt ccgcgatgca tccccggcgt tttgttctat caccgctgag cgtgccatca 60 aggtcgaact ggccaccggt ggcgccgtca ctcgcttcga gctcaggccg gacatcgatt 120 ggggcggcct gccggctggc agcgttgccc ccgaccttga gcaaatcgta gctgcaccga 180 gcgtgatggt gcagggtgtc ggtagtgctg ttcaggcatc cagtgccgag gtggcgccgt 240 gactttcctt tcgaagatgg ctcgcttcct cgggctggat gttcagcgtg atcggatcgc 300 ggccgcctgg cgcgggcagg gctttgaggc tggtgtcatt tacgacgaag cagagattct 360 gcggcgtctt ggctggcgtg aacagcttca acggcgactt cgacaagttc tccggtggtt 420 cggtttatca gcatcgccat cccgttgtcg tctagggcgc cgcgcaccga gtccccaatc 480 cgaagctcgc caaccagcac atcaacgatc gcaaacccat cgtcggtgcg aatcgcatag 540 cgaggaaggt tagcgtgata gccgcatacc actcccatta ctgcgatacc tcg 593 <210> 57 <211> 155 <212> DNA <213> Pseudomonas aeruginosa <400> 57 cacccttggc gtcgccttgc ctgctcgtac ttggtgagga tgtggccgaa cagttccggg 60 ctgaccgaag agaagtaggt cggcggaacg ttctcgctcg gcttgaccag ttccggatag 120 gtgccgatcg acaggctggc cggtgccgcc ttgac 155 <210> 58 <211> 114 <212> DNA <213> Pseudomonas aeruginosa <400> 58 cgcccttcgc tgaccgccgg gctggacggt gcgatcaacc gcgccaaggt cgatcccgcc 60 cgcgccgagc aggcccgacg catgcagcag gcggcccagc agcagatgca gcaa 114 <210> 59 <211> 67 <212> DNA <213> Pseudomonas aeruginosa <400> 59 cgccctttgc ccctatgcgc tctactacat ccgcaaggga atcgctaagg tacagtccga 60 ggcggag 67 <210> 60 <211> 602 <212> DNA <213> Pseudomonas aeruginosa <400> 60 atggcctggg gcatgggaaa gagtagccta gcgtgcccct gcgcattgag aaaggggaga 60 agtgccagcg acgcgctgcg gatcatgcag ccgatacttg tgtacaggcc gatcacgcag 120 atcgcaccga ccgcgcctgt ccggctgacc atccctgggc acgggttgcc tggcacgtgg 180 ctggcctggg ctgatggtgt ccagggcatg cccgaactga accgcgctcg acttcggcaa 240 ttgcctcacc gtgtcgcgtc catcgacgac gacacgatcg agatcaacct gctgtcagcc 300 gttgggctgg cgcctgttgg cggacagttg atctaccagc cacctgttga cctggctggc 360 gccgaggtac gaatgcagat ccgcgaagcg ccaggcggga ctgtgctgat gacgctggcg 420 ctcggctctg gcctggatct cgccggcgcc ggaacgatct cgcgggagat atcggcctcc 480 gctacctcgg agctgatgtg gtcgtcggcg gtctacgacc tggacgtgac atacccggat 540 ggaacggtcc accgctatta cagcgggccg atcagtgtga gccatggggg agggtgctat 600 gg 602 <210> 61 <211> 482 <212> DNA <213> Pseudomonas aeruginosa <400> 61 taacagcttt ctgatcaatc ctcagcctgt aacgtgaccg tcataggagg gacgtcgcca 60 agccctacct gttccagggg agggtatgag cgagacctcc gtaaagccag ggtccagggc 120 aaggaagttc ccggcatgtc cagagaagag gtggaaagca tttatgggaa ggcgaaccgg 180 aatggcagca ctgctggtgc cggggccgtt acctactgga atgacaagta catcgatcag 240 accacggttt ctttcgaccg aaacggttgc gttcaaggct cataccagtc gggccacaaa 300 aactgatcca ccgtcgtcaa tcaccccgct ccggcggggt ttttattttg gagccctcaa 360 tcatgaagcc tgcctgcgtg cccctgcgca ttgagaaagg ggcgacgttc cgcgacgcgc 420 tgcgtttcat gtagccgata cttgtgtaca ggccgatcac gcagatcgca ccgaccgcgc 480 ct 482 <210> 62 <211> 646 <212> DNA <213> Pseudomonas aeruginosa <400> 62 tctcgcccca gtgtaggcgt ggcttcatat gcggtaagtc ctcgtaccat aatgttgaag 60 gaaaaggcta gccgaccgaa catttccctg tgcgtcatac cacccctgtg atcaatgccg 120 aggtctgagt ccactcgtca cgataactcg ccgtcacaac agggtcctca ggaaggctgg 180 actcatcgat atgcacgtga gcagggcttc ccatcgcttg accgcgcgtc tcaatgacgg 240 tcattgtcag caccttggat cgatccgcat caggatctcg tatcgacgga gagacagtga 300 tttcaatcaa tccatagaag cccacgggag caccggaatt cgatgagcca ctaatccacg 360 atgttccagg cggttgctca attggtgaca aatcggtagt tcgaacgtaa acaccgaaaa 420 ggagacgatt ttgatagacc ccaagcattg atagctcgct caacacaact ttgttcgtca 480 gttgccagcc gtcaaatgaa acatcttttg ccctgacagc gcatgctggt tgttcttctc 540 cctgaccaat taaatctgca cccaagtcga gcgtggccat ggtgttgagg tcatttccaa 600 cccataggcg aaactgcggt gtacctgcct cgtcccctta aatcat 646 <210> 63 <211> 90 <212> PRT <213> Pseudomonas aeruginosa <400> 63 Leu Asn His Phe Gly Met Gln Pro Lys Ala Ser Ile Gly His Asn Ile 1 5 10 15 Val Phe Ala Ala Glu Val Leu Asp Gly Val Phe Lys Asp Gly Val Gly             20 25 30 Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp Ala Arg Val Val Ala         35 40 45 His Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser Ser Leu Thr Leu Asp     50 55 60 Pro Ala Lys Leu Ala Asp Gly Asp Tyr Lys Phe Ala Cys Thr Phe Pro 65 70 75 80 Gly His Gly Ala Leu Thr Ser Ser Gln Val                 85 90 <210> 64 <211> 95 <212> PRT <213> Neisseria gonorrhoeae <400> 64 Leu Lys His Thr Gly Thr Gln Pro Lys Ala Ser Met Gly His Asn Leu 1 5 10 15 Val Ile Ala Lys Ala Glu Asp Met Asp Gly Val Phe Lys Asp Gly Val             20 25 30 Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp Ala Arg Val Val         35 40 45 Ala His Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser Ser Leu Thr Leu     50 55 60 Asp Pro Ala Lys Leu Ala Asp Gly Asp Tyr Lys Phe Ala Cys Thr Phe 65 70 75 80 Pro Gly His Gly Ala Leu Met Asn Gly Lys Val Thr Leu Val Asp                 85 90 95 <210> 65 <211> 95 <212> PRT <213> Neisseria meningitidis <400> 65 Leu Lys His Thr Gly Thr Gln Pro Lys Ala Ser Met Gly His Asn Leu 1 5 10 15 Val Ile Ala Lys Ala Glu Asp Met Asp Gly Val Phe Lys Asp Gly Val             20 25 30 Gly Ala Ala Asp Thr Asp Tyr Val Lys Pro Asp Asp Ala Arg Val Val         35 40 45 Ala His Thr Lys Leu Ile Gly Gly Gly Glu Glu Ser Ser Leu Thr Leu     50 55 60 Asp Pro Ala Lys Leu Ala Asp Gly Asp Tyr Lys Phe Ala Cys Thr Phe 65 70 75 80 Pro Gly His Gly Ala Leu Met Asn Gly Lys Val Thr Leu Val Asp                 85 90 95 <210> 66 <211> 96 <212> PRT <213> Pseudomonas aeruginosa <400> 66 Leu Ser His Pro Gly Asn Leu Pro Lys Asn Val Met Gly His Asn Trp 1 5 10 15 Val Leu Ser Thr Ala Ala Asp Met Gln Gly Val Val Thr Asp Gly Met             20 25 30 Ala Ser Gly Leu Asp Lys Asp Tyr Leu Lys Pro Asp Asp Ser Arg Val         35 40 45 Ile Ala His Thr Lys Leu Ile Gly Ser Gly Glu Lys Asp Ser Val Thr     50 55 60 Phe Asp Val Ser Lys Leu Lys Glu Gly Glu Gln Tyr Met Phe Phe Cys 65 70 75 80 Thr Phe Pro Gly His Ser Ala Leu Met Lys Gly Thr Leu Thr Leu Lys                 85 90 95  

Claims (32)

(a) a nucleic acid selected from SEQ ID NOS: 26-62, and (b) an isolated nucleic acid molecule comprising a polynucleotide sequence selected from sequences that are at least 90% homologous to the nucleic acid selected in SEQ ID NOS: 26-62. The nucleic acid molecule of claim 1, wherein the polynucleotide is SEQ ID NO: 26. A pharmaceutical composition comprising the isolated nucleic acid sequence of claim 1 and a pharmacologically acceptable carrier. 4. The pharmaceutical composition of claim 3, further comprising at least one cupredoxin peptide. 4. A pharmaceutical composition according to claim 3, which is formulated for intravenous administration. 4. A pharmaceutical composition according to claim 3, which is formulated for subcutaneous administration. 4. A pharmaceutical composition according to claim 3, which is formulated for topical administration. The method of claim 4, wherein the cupredoxin peptide is Pseudomonas aeruginosa ), Alcaligenes faecalis ), Achromobacter xylosoxidan ), Bordetella bronchiseptica , Mcthylomonas sp . ), Meningitis bacteria (Neisseria meningitidis), yeomgyun (Neisseria gonorrhea ), Pseudomonas fluorescens , Pseudomonas chlorolapis chlororaphis ), Silyla Pastidiosa ( Xylella) fastidiosa ), enteritis Vibrio ( Vibrio parahaemolyticus ) pharmaceutical composition, characterized in that it is derived from an organism selected from. 5. The cupredoxin peptide according to claim 4, wherein the cupredoxin peptide is azurin, pseudoazurin, platocyanin, rusticyanin, Laz, auracyanin, stelaci Pharmaceutical composition, characterized in that some or all of the protein selected from, stellacyanin, cucumber basic protein (cucumber basic protein). The pharmaceutical composition of claim 4, wherein the cupredoxin peptide comprises some or all of the peptides selected from SEQ ID NOS: 1-25. A method of treating a patient, wherein the patient consists of co-administering the pharmaceutical composition according to claim 3 and the cupredoxin peptide. The method of claim 11, wherein the patient suffers from the disease. The method of claim 12, wherein the patient suffers from a cancer disease. A method of treating a patient, said method comprising the administration of the pharmaceutical composition according to claim 4 to the patient. The method of claim 14, wherein the patient suffers from the disease. The method of claim 15, wherein the patient suffers from a cancer disease. The method of claim 11, wherein the pharmaceutical composition is administered by a route selected from intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection and oral. 18. The method of claim 17, wherein the method of administration is intravenous. 12. The method of claim 11, wherein the additional prophylactic or therapeutic drug is co-administered. A cell having a heterozygous gene for azurin derived from Pseudomonas aeruginosa, wherein the cell expresses azurin upon contact with cancer cells. The cell of claim 20, wherein in the heterologous gene for azurin, the coding sequence of the azurin is replaced with the coding sequence for the target protein and the cell expresses the target protein upon contact with the cancer cell. A Pseudomonas aeruginosa cell having a coding sequence of azurin in its genome, wherein the coding sequence of the azurin is replaced with a coding sequence for the target protein, wherein the cell expresses the target protein upon contact with the cancer cell. The cell of claim 21, wherein the target protein is selected from prophylactic, therapeutic, cytotoxic and diagnostic proteins. A method of treating a patient, the method comprising administering a cell according to claims 20-23. The method of claim 24, wherein the patient suffers from the disease. The method of claim 25, wherein the patient suffers from a cancer disease. The method of claim 26, wherein the cancer is selected from melanoma, breast, pancreas, glioblastoma, astrocytoma, lung, rectal cancer, neck, head, bladder, prostate, skin and neck cancer. A method of diagnosing cancer in a patient, the method comprising administering a cell according to claim 21 wherein the cell expresses a diagnostic target protein. The method of claim 24, wherein the pharmaceutical composition is administered by a route selected from intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection and oral. The method of claim 29, wherein the method of administration is intravenous injection. The method of claim 22, wherein the target protein is selected from prophylactic, therapeutic, cytotoxic and diagnostic proteins. The method of claim 28, wherein the pharmaceutical composition is administered by a route selected from intravenous injection, intramuscular injection, subcutaneous injection, inhalation, topical administration, transdermal patch, suppository, vitreous injection and oral.

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